Equal Aliquots Research Articles

B-310 Freezing and thawing serum without mixing prior to analysis causes false low Thyroglobulin results

Abstract Background Thyroglobulin (Tg), a glycoprotein that is synthesized in follicular cells of the thyroid gland, is the precursor of Thyroxin (T4) and Triiodothyronine (T3). Tg is a biomarker used for many clinical conditions, including monitoring for recurrence in patients with differentiated thyroid carcinoma. Typically, Tg levels drop to very low or undetectable levels after surgical excision and/or radioiodine ablation of the thyroid, with post-operative levels greater than 1-2 ng/ml indicating potential disease recurrence. Discordant Tg results in a patient sample were identified during a quality check of Tg results. This study investigates the root of cause of the discrepant Tg results. Given that: 1) Tg analyses were performed twice a week at our laboratory due to low daily test volume and patients’ samples received on an unanalytical day were stored at -20°C until analysis; 2) Tg analyses were performed by various medical technologists; 3) sample mixation prior to analysis was not routine practice for our automated laboratory, we hypothesized that the discrepancy was due to inconsistent sample handling, e.g., mixation vs unmixation prior to analysis. Methods Analysis of the daily QC and PT results in the period of the incident occurence ruled out causes in analytical phase. We split 52 de-identified patient serum samples into two equal aliquots and subsequently stored both aliquots at -20°C for 48 hours. Both aliquots of each sample were thawed at room temperature for 1 hour. One of the aliquots was analyzed for Tg without mixing, and the other was analyzed after mixing using a Vortexer for 4 seconds at 250 xg. To investigate if Tg concentrations form a gradient from the top to the bottom of a red-top vacutainer tube, five patient samples with Tg at 2.59, 2.79, 2.92, 52.37 and 144.12 ng/mL were thawed without mixing following storage at -20°C for 48 hours. Five layer (600 μl/layer) aliquots were carefully and sequentially taken from top to bottom for each specimen, and the Tg concentration in the aliquot of each layer was determined using the Beckman Coulter Access-2 Immunoassay. Results Of the 52 patients samples, 14 Tg results were excluded from statistical analysis due to exceeding the assay dynamic range (2 above and 12 below the upper and low limits, respectively). Analysis of the remaining 38 patient samples showed the mean of Tg concentrations was 22.8% (SD 18%) higher in mixed samples than the un-mixed samples. Regression analysis showed an R2 = 0.9976 (Mixed Tg = 1.1523 x Un-mixed + 0.4174, range 0.1 - 275.7 ng/mL, intercept 95%CI -0.721 to 1.556, slop 95%CI 1.1331 to 1.1715, p-value <0.001). Results of Tg gradient analysis showed significant higher Tg values in the 2nd, 3rd, 4th and 5th than the 1st layer by 26% (SD 11%), 63% (26%), 112% (57%), and 160% (76%), respectively. Conclusions False Tg values were found in samples without mixing before analysis after thawing from frozen state, due to formation of Tg concentration gradient in test tube. For obtaining reliable results, samples must be mixed before analysis when thawed from frozen storage.

Effect of 532nm and 589nm Diode-Pumped Solid-State (DPSS) Lasers with Different Doses on Concentration of Total Protein and Albumin in Vitro.

To test the effects of Low-level laser irradiation at 532nm and 589nm with different doses and exposure times on total protein and albumin in blood plasma. This experimental study was conducted from November 2021 to April 2022 at Mustansiriyah University, Baghdad, Iraq, and comprised blood samples from healthy adults. The samples were stored in tubes containing anticoagulant ethylenediaminetetraacetic acid. Photoradiation was managed using green laser 532nm and yellow laser 589nm diode-pumped solid-state lasers with different doses. For total protein test and albumin test, each sample was divided into 3 equal aliquots. The first was control without radiation, the second was irradiated by a laser at 532nm, and the third was irradiated at 589nm with 30J/cm², 50J/cm², 70J/cm² and 90 J/cm² doses of irradiation. All samples were examined by using a spectrophotometer device. Data was analysed using SPSS 25. There were 24 samples from healthy adults in all.The most significant difference in total protein concentration occurred at dose 70J/cm² for both wavelengths compared to the controls (p<0.05). When this optimal dose was applied to the albumin test, a clear decrease in the albumin concentration was found for both 532nm and 589nm lasers (p<0.05). At wavelengths of 532nm and 589nm, the laser light induced processes that caused a reduction in total protein and albumin.

Evaluation of the effect of yellow laser (DPSS) light on human lymphocytes viability in vitro.

To evaluate the lymphocyte apoptotic impact of yellow laser light in vitro. The experimental study was conducted from November 2021 to April 2022 at the Postgraduate Medical Physics Laboratory, Mustansiriyah University, Baghdad, Iraq, and comprised blood samples from healthy volunteers. The samples were subjected to 1:1 dilution in isotonic phosphate buffered saline, and each sample was divided into two equal aliquots; one for irradiation, and other as control.The percentage of apoptotic lymphocyte was estimated before and after radiation exposure with low-level laser 589nm at 30J/cm², 50J/cm² and 70J/cm² energy intensities. Post-exposure evaluation was done immediately, and 1 and 2 hours after radiation on the viability of normal human cells. The vitality of the cells was determined using the trypan blue exclusion test. Data was analysed using SPSS 24. There were 26 healthy volunteers (16 male, 10 females) with an age range of (20-40) years. After the exposure, the percentage of lymphocytes that apoptose increased significantly when cells were irradiated at 70J/cm2 immediately compared to the controls (p≤ 0.05), but there was no significant differences with the 30J/cm² dose. After 1 and 2 hours, a significant reduction in the proportion of apoptotic lymphocytes was observed (p<0.05). Yellow low-level laser showed a noticeable protective effect on lymphocytes by decreasing the percentage of dead cells.

A comparison study of 589 and 650 nm on improving stored whole blood stability by irradiation with a low-power diode pumping solid-state laser.

To assess the effects of diode-pumped solid-state laser irradiation with 589nm and 650nm wavelengths on the stability of stored red blood cells in vitro. This is an intervention study that was conducted from April to July 2021 at the Physiology and Medical Physics Laboratory, College of Medicine, Mustansiriyah University, Baghdad, Iraq, and comprised samples of healthy, adult human blood that were put in tubes with citrate-phosphate dextrose-adenine as an anticoagulant. The blood sample was divided into eight equal aliquots and stored for 21 days at 4ºC. The stability test was done on days 0, 7, 14 and 21 of storage time for non-irradiated and radiated aliquots. For 15 minutes, the irradiated aliquots were subjected to a diode-pumped solid-state laser with a wavelength of 589nm or a laser with a wavelength of 650nm at frequencies of 30, 50 and 70 J/cm². Data was analysed using SPSS 24. Exposure of the whole blood to 589nm and a radiation dose of 70J/cm², 50J/cm² and 30J/cm² was associated with a significant reduction in the percentage of haemolysis that ranged 23-42% throughout the whole storage time. Exposure of the whole blood to 650nm wavelength low-level laser therapy and a radiation dose of 70J/cm², 50J/cm² and 30J/cm² was associated with much less reduction in the percentage of haemolysis that ranged 5-9% throughout the whole storage time. Both 589nm and to some extent 650nm low-level laser irradiation generally reduced haemolysis. The wavelength of 589nm lasers had a more effective influence than the 650nm counterpart in improving the stored red blood cells.

The effect of a low-level yellow laser on improving the stability of stored whole blood.

To explore if low-level laser 589nm may stabilise stored whole blood and red blood cell suspension in vitro. The interventional study was conducted from November 2021 to April 2022 at the Postgraduate Medical Physics Laboratory, Mustansiriyah University, Baghdad, Iraq, and comprised blood samples from healthy adults. The samples were obtained put in tubes containing anticoagulant citrate-phosphate dextrose-adenine. Each sample was divided into eight equal aliquots and stored for 21 days at 4ºC. Of the 8 aliquots, 4 were takebn as controls, while the other 4 were irradiated by 589nm laser beam with a radiation dose of 50J/cm² for 15min. Stability test measured by the percentage of haemolysis of an overnight stored red blood cell in saline solution was done on days 0, 7, 14 and 21 for non-irradiated and radiated aliquots. Data was analysed using SPSS 24. Eight subjects (4 males and 4 females) were included, with mean age 21±3 years (range: 19-24 years). Whole blood samples showed a significant reduction in haemolysis by 40%, 20% and 42% compared to the control samples at days 0, 7 and 14, respectively (p<0.05). Red blood cell suspension showed a significant reduction in haemolysis by 9% compared to the control samples only at day 21 (p<0.05). Low-level laser 589nm beam at 50J/cm² radiation dose was more effective on the stability of stored whole blood than the red blood cell suspension.

Effect of different concentrations of inulin on ram sperm quality during cryopreservation

BACKGROUND: In reproductive biotechnology, sperm cryopreservation has a vital role to play. Cryopreservation of sperm produces reactive oxygen species (ROS), which disrupt sperm function and structural competence. Numerous protective chemicals, including fructans, have been used during sperm cryopreservation. OBJECTIVES: To evaluate the effect of different concentrations of the fructosan inulin on ram sperm quality parameters, superoxide dismutase (SOD) activity, and malondialdehyde (MDA) production after freezing and thawing. MATERIALS AND METHODS: The pooled samples from four healthy rams were divided into seven equal aliquots and diluted in a Tris-base extender supplemented with 1, 2, 4, 8, 16, and 28 mM of inulin or without inulin supplementation (control). By using liquid nitrogen vapor, the semen was frozen and stored at 196°C. RESULTS: The total motility, viability, and DNA integrity were significantly improved after freezet-hawing with 28 mM inulin, compared to other treatment groups (P&lt;0.05). A Tris-based extender containing 16 and 28 mM of inulin displayed the highest levels of ram sperm membrane integrity when compared with the control (p&lt;0.05). The abnormality of ram sperm was increased during freeze-thawing at control and 1 mM of inulin, compared to 16 and 28 mM of inulin (P&lt;0.05). Additionally, 28 mM of inulin decreased MDA and increased SOD activity in ram sperm in comparison with the other treatments (P&lt;0.05). CONCLUSION: As a result, 28 mM of inulin could be beneficial for the cryopreservation industry and reduce the harmful effects of freeze-thawing on ram sperm.

Effect of cooling rates and equilibration times on post-thaw sperm quality of Kail rams

Context The conflicting findings regarding the impact of equilibration time on post-thawed sperm quality underscore the need for further research to evaluate the impact of equilibration time and cooling rate on post-thaw sperm quality of ram semen. Aims The current study aimed to assess the combined impact of cooling rates and pre-freezing equilibration times on post-thaw sperm quality in Kail ram semen (n = 5). Methods Semen collection was performed using an artificial vagina at 42°C. The pooled semen was divided into equal aliquots and subjected to either slow cooling (SC, −0.27°C/min) or moderate cooling (MC, −0.36°C/min) rates, transitioning from 37°C to 4°C. Equilibration times of 0, 4, 8, and 12 h were employed before freezing. Key results Semen samples undergoing the SC rate and equilibrated for 4 h exhibited higher (P &lt; 0.05) percentages of progressive motile (PM), rapid progressive (RP), and medium progressive (MP) sperm compared with the MC rate. However, total motility remained unaffected by the cooling rate (P &lt; 0.05). Semen equilibrated for 4 h demonstrated higher (P &lt; 0.05) percentages of PM and RP sperm, as well as improved kinematics (curvilinear velocity, average path velocity, and straight-line velocity) compared with other equilibration times. Nevertheless, equilibration time had no (P &gt; 0.05) impact on the amplitude of the lateral head displacement for semen samples subjected to the MC rate. Notably, the cooling rate did not affect post-thaw sperm kinematics, plasma membrane integrity, or live-sperm percentage (P &gt; 0.05). Semen samples equilibrated for 4, 8, and 12 h exhibited a higher (P &lt; 0.05) percentage of sperm with intact plasma membrane and viability than did those equilibrated for 0 h. Conclusions In conclusion, slow cooling rate and a 4 h equilibration period were shown to be optimal for preserving post-thaw sperm quality in Kail rams. Implications The findings highlighted the combined effect of cooling rate and equilibration time on post-thaw sperm quality for optimising sperm cryopreservation protocols in the context of ram semen.

The effects of MitoQ as a mitochondrial-targeted antioxidant in a plant-based extender on buck sperm quality parameters during cryopreservation

Sperm cryopreservation plays an important role in the artificial insemination (AI) industry of small ruminants. It, however the use of frozen-thawed goat semen is limited due to the insufficient number of sperm with good biological functions. Mitochondria are the most sensitive organelles to cryopreservation damage in sperm. This study was conducted to determine the effects of MitoQ, the mitochondrial-targeted antioxidant, in a plant-based extender on the quality parameters of Markhoz goat sperm after the freezing and thawing process. Semen samples were collected and diluted in the extender, divided into five equal aliquots and supplemented with 0, 1, 10, 100 and 1000 nM MitoQ and cryopreserved in liquid nitrogen. After thawing, sperm motility, membrane functionality, abnormal morphology, mitochondrial activity, acrosome integrity, lipid peroxidation (LPO), DNA fragmentation, reactive oxygen species (ROS) concentration, viability and apoptotic-like changes were measured. The use of 10 and 100 nM MitoQ resulted in higher (P≤0.05) total motility (TM), progressive motility (PM), viability, membrane functionality, mitochondrial activity, and acrosome integrity compared to the other groups. On the other hand, LPO, apoptotic-like changes, DNA fragmentation and ROS concentration were lower (P≤0.05) in MQ10 and MQ100 groups compared to the other groups. MitoQ has no effect (P>0.05) on sperm abnormal morphology and velocity parameters. In conclusion, MitoQ can reduce oxidative stress by regulating mitochondrial function during the cryopreservation process of buck sperm and could be an effective additive in the cryopreservation media to protect sperm quality.

Tissue oxygen saturation is positively correlated with oxygen delivery and cardiac output in a canine hemorrhagic shock and resuscitation model.

To determine if tissue oxygen saturation (StO2) correlates with oxygen delivery (DO2) and/or cardiac output (CO) in a canine hemorrhagic shock model. 8 healthy purpose-bred dogs. Dogs were anesthetized, and hemorrhagic shock was induced by withdrawing up to 60% of total blood volume, targeting a mean arterial pressure (MAP) of 40 mm Hg. The withdrawn blood was returned to the patient in 2 equal aliquots. Data was collected at 4 time points: 10 minutes after MAP was stabilized under anesthesia (time point [TP]-1), 10 minutes after up to 60% of blood volume was removed to target a MAP of 40 mm Hg (TP2), 10 minutes after the return of 50% of shed blood (TP3), and 10 minutes after the return of the remaining 50% of shed blood (TP4). Total blood volume withdrawn, StO2, CO, heart rate, and MAP were recorded, and DO2 was calculated at each TP. Mean StO2 significantly decreased between TP1 (77.8% [± 9.54]) and TP2 (44.8% [± 19.5]; P < .001 vs TP1). Mean StO2 increased to 63.1% (± 9.85) at TP3, but remained significantly lower compared to TP1 (P = .002). There was no difference between mean StO2 at TP4 (82.5% [± 12.6]) versus TP1 (P = .466). StO2 has a strong, positive correlation to both CO (r = 0.80; P < .001) and DO2 (r = 0.75; P < .001). A decrease in StO2 may be used in conjunction with physical examination findings and diagnostic parameters to support a diagnosis of shock. The return of shed blood was correlated with increases in StO2, DO2, and CO, suggesting that StO2 may be used as a marker of adequate resuscitation.

Camellia oil with its rich in fatty acids enhances post-thawed boar sperm quality

BackgroundBoar sperm are highly susceptible to specific conditions during cryopreservation, leading to a significant decrease in their fertilizing potential due to damage to their membranes. Camellia oil, known for its fatty acids with antioxidant and biological properties, has not been previously explored for the cryopreservation of boar semen. This study aimed to examine the effects of camellia oil on post-thawed boar sperm quality. Boar semen ejaculates (n = 9) were collected and divided into six equal aliquots based on camellia oil concentrations (0, 0.5, 1, 1.5, 2 and 2.5% v/v) in the freezing extender. Semen samples were processed and cryopreserved using the liquid nitrogen vapor method. Thereafter, frozen semen samples were thawed at 50 °C for 12 s and evaluated for sperm morphology by scanning electron microscope, sperm motility using a computer-assisted sperm analyzer, sperm viability, acrosome integrity, mitochondrial function, MDA level and total antioxidant capacity.ResultsThe results demonstrated that the supplementation of 1.5% (v/v) camellia oil showed superior post-thaw sperm qualities such as improved sperm morphology, motility, acrosome integrity and mitochondrial function by 14.3%, 14.3% and 11.7%, respectively, when compared to the control group. Camellia oil at a concentration of 1.5% (v/v) showed the lowest level of MDA (18.3 ± 2.1 µmol/L) compared to the other groups.ConclusionsIn conclusion, adding 1.5% (v/v) camellia oil in the freezing extender reduced the oxidative damage associated with cryopreservation and resulted in a higher post-thawed sperm quality.

A simple microplastic splitter for subsampling expanded polystyrene particles

With the high number of microplastic-like particles captured by net hauls including manta or neuston nets, it is often required to subsample in order to decrease sample volume for microplastic enumeration and analysis. Plankton splitter is commonly used to divide microplastic samples. However, current devices such as Folsom plankton splitter and Motoda box splitter have accuracy issues in separating highly buoyant microplastics, namely expanded polystyrene (EPS) as they tend to adhere to the inner walls. Inspired by an apple cutter, we have developed a simple radial splitter made of stainless steel that efficiently divides EPS microplastic samples into precise aliquots. With this simple device, we uniformly divided EPS microplastic samples from marine environments into eight aliquots with no significant differences. The device is a versatile tool to partition all buoyant microplastics including polypropylene and polyethylene microplastics.•The method developed facilitates the precise division of buoyant microplastics into equal aliquots.•The method is specifically effective in splitting expanded polystyrene particles with high buoyancy.

Improving chilled and frozen buck sperm characteristics by adding melatonin and L-carnitine to the preservation medium.

This study evaluated the effects of melatonin (MLT) and L-carnitine supplementation on sperm quality and antioxidant capacity during chilled and cryopreservation. Twenty-four ejaculates were collected from six Damascus bucks, 4 ejaculates each, from mid-September to mid-October 2022. The pooled semen from each collecting session was divided into 5 equal aliquots after being diluted (1:10) with Tris-citric acid egg yolk extender. The first aliquot served as a control (treatment-free). MLT was added to the second and third aliquots at low and high doses (LD: 4 and HD: 8 μL/mL) (v/v), respectively, while L-carnitine (LC) was added to the fourth and fifth aliquots at the same aforementioned doses. The aliquots were stored at 4°C for 48 h to assess sperm physical and morphological characteristics, alongside lipids peroxidase (LP) production and glutathione peroxidase (GPX) activity. The optimum doses of MLT and LC that showed potential for maintaining sperm characteristics throughout the chilled storage period were further investigated for protecting the spermatozoa after exposure to cryopreservation stress compared to the control. The results showed higher sperm motility (%) in the MLT-HD group, higher (p ≤ .05) sperm viability (%) in the MLT-LD, and both aliquots of LC at T24 hours of chilled preservation. Normal sperm (%) was higher (p ≤ .05) in both LC-LD and MLT-LD groups than other groups, while sperm acrosome integrity (%) was higher (p ≤ .05) in the LC-LD group. Morphological abnormalities (%) were improved (p ≤ .05) in all treated aliquots compared with control. The mean value of GPX activity was higher (p ≤ .05) in both MLT groups, while the concentration of LP increased (p ≤ .05) in the LC-HD or control groups. Furthermore, supplementing buck sperm medium with 4 μL/mL of MLT or LC improved (p < .05) the sperm characteristics and decreased (p < .05) DNA fragmentation index after thawing.

Granulocyte-Macrophage Colony-Stimulating Factor Cytokine Addition After the Freeze-Thawing Process Improves Human Sperm Motility and Vitality in Asthenoteratozoospermia Patients.

The cryopreservation-thawing process of spermatozoa cells has negative impacts on their structure, function, and fertility parameters, which are known as cryoinjury. Asthenozoospermia patients are more susceptible to cryoinjury. Granulocyte-macrophage colony-stimulating factor (GM-CSF) increases sperm glucose uptake via the induction of glucose transporters, resulting in increased sperm motility. This study aimed to investigate the efficiency of GM-CSF supplementation of the cryopreservation media for semen samples of asthenoteratozoospermia patients. The study was carried out on 20 semen samples from infertile men referred to diagnosing semen analysis. To avoid subjective bias, two main sperm motility parameters, including velocity along the curvilinear path and velocity along the straight-line path were considered by the computer-assisted sperm analysis system. Afterward, each semen sample was divided into three equal aliquots and randomly assigned to one of the following groups: group I (control, freezing media only), group II (+GM-CSF, freezing medium supplemented with 2 μL/mL GM-CSF), or group III (GM-CSF added after thawing and washing). Following semen thawing, standard parameters, mitochondrial membrane potential (MMP), and the DNA Fragmentation Index were analyzed. Total sperm motility (progressive and non-progressive) improved significantly in group III samples after a 30-minute incubation with GM-CSF compared with the control group (26.5% ± 3.1% vs. 17.51% ± 2.59%). However, no differences in progressive motility or sperm morphology were found among the three thawed samples. The percentage of vitality was significantly higher in group III compared with the other two groups (28.38% ± 3.4% vs. 22.4% ± 3.08% and 22.14% ± 2.77%, respectively) (p < 0.05). JC-1 levels (a marker of MMP) were not significantly different between the examined groups (44.95% ± 8.26% vs. 36.61% ± 6.95% vs. 46.67% ± 7.7%, for control, group II, and group III, respectively) (p > 0.05). GM-CSF may be advantageous as an additive after freezing, improving total motility and viability after 30 minutes of post-thaw incubation; however, when supplied to the freezing media before cryopreservation, it is unable to protect against cryoinjury.

A-111 There is More to Diluting Icterus Interference than Meets the Eye

Abstract Background Total protein is measured in serum and body fluids as part of routine evaluations of general nutritional status and differentiating exudative and transudative effusions. Spectral interference thresholds for bilirubin (e.g., icterus) is set by assay manufacturers to prevent inaccurate results from being reported. The Roche Cobas serum total protein assay package insert states no significant interference from bilirubin up to an icterus (I) index of 20. Body fluids tend to have lower total protein concentrations; therefore, thresholds were derived during prior validation studies and set to I = 10, a threshold that is exceeded routinely. Laboratories can mitigate some interferences by recollecting (e.g., hemolysis) or ultracentrifuging (e.g., lipemia) specimens. Serial dilutions are another option used; however, it is imperative that the laboratory understands the mechanism of interference and can demonstrate that it is mitigated by sample dilution. The aim of this study was to investigate whether bilirubin interference can be mitigated by sample dilution in pleural fluid, peritoneal fluid, and serum specimens. Methods Residual clinically ordered pleural fluid (n = 3), peritoneal fluid (n = 3), and serum (n = 3) samples submitted for protein testing were split into two equal volume aliquots with concentrations ranging 2.7 to 4.3 g/dL in pleural fluids, 3.9 to 5.4 g/dL in peritoneal fluids, and 3.8 to 11.0 g/dL in serum. One aliquot was spiked (&amp;lt;10% by volume) with a bilirubin conjugate solution (∼1000 mg/dL) to surpass the icterus index threshold (spiked). The second aliquot was spiked with an equal volume of 0.9% saline (control). Total protein was measured on the Roche Cobas c701 instrument in both samples to calculate difference (Spiked-Control). The mean(SD) difference was calculated for each sample type. Each spiked sample was serially diluted (2-fold, 4-fold and 8-fold) with 0.9% saline. Total protein was measured for each diluted sample. Mean(SD) difference was calculated (DilutedX-Control), where X = 2-fold, 4-fold, 8-fold. Linear regression analysis was performed by plotting the measured (x-axis) vs expected (y-axis) total protein concentration. Slope, y-intercept, and R2 were determined from serially diluted samples where the expected concentrations were derived using the spiked concentration to replicate the practice of diluting an icteric sample. Results Bilirubin spiking (average icterus index = 33.5) into pleural fluid, peritoneal fluid, and serum, respectively demonstrated mean(SD) bias (g/dL) = −1.0(0.2), −0.9(0.0), and −0.7(0.0). Subsequent 2-fold dilution demonstrated mean(SD) bias (g/dL) = −1.0(0.2), −1.0(0.0), and −0.8(0.1), 4-fold dilution with mean(SD) bias (g/dL) = −1.1(0.2), −1.0(0.0), and -1.0(0.1), and 8-fold dilution with mean(SD) bias (g/dL) = −1.3(0.2), −1.0(0.0), and −1.4(0.1). Diluting pleural, peritoneal, and serum samples, respectively spiked with bilirubin resulted in slope = 0.99, 1.00, and 0.99; y-intercept = 0.04, 0.01, and 0.07; R2 = 0.99, 0.99, 0.99. Conclusion The dilutions exhibited a linear dilution response, however the dilution results never returned to baseline (non-spiked) concentration. Laboratories should exercise caution when conducting serial dilutions to diminish bilirubin interference in samples when measuring total protein as the mechanism of interference does not appear to be reversible by dilution. This behavior was demonstrated in both body fluids and serum.

Palm Kernel Meal Protein Hydrolysates Enhance Post-Thawed Boar Sperm Quality.

Boar sperm is sensitive to particular conditions during cryopreservation, resulting in an extreme reduction in fertilizing ability due to damage to the sperm membranes. PKMPH contains bioactive peptides that have antioxidant and antimicrobial activities. There is no information on the use of palm-kernel-meal-derived bioactive peptides for boar semen cryopreservation. This study aimed to examine the effects of bioactive peptides from PKMPH on post-thawed boar sperm quality. Boar semen ejaculates (n = 17) were collected and divided into six equal aliquots based on PKMPH concentrations (0, 1.25, 2.5, 5, 10, and 15 µg/mL) in a freezing extender. Semen samples were processed and cryopreserved using the liquid nitrogen vapor method. Thereafter, the frozen semen samples were thawed at 50 °C for 12 s and evaluated for sperm motility using a computer-assisted sperm analyzer and for sperm viability, acrosome integrity, mitochondrial function, and lipid peroxidation by measuring the level of malondialdehyde (MDA). The results demonstrate that the supplementation of PKMPH with 2.5 µg/mL afforded superior post-thawed sperm qualities, such as increased total motility, viability, acrosome integrity, and mitochondrial function by 10.7%, 12.3%, 18.3%, and 12.7%, respectively, when compared to the control group. PKMPH at a concentration of 2.5 µg/mL showed the lowest level of MDA (40.6 ± 2.0 µMol/L) compared to the other groups. In conclusion, adding PKMPH peptides at 2.5 µg/mL to the freezing extender reduced the oxidative damage associated with cryopreservation and resulted in higher post-thawed sperm quality.

Preservation of the quality and fertility potential of post-thawed rooster sperm using MitoQ

Cryopreservation of rooster spermatozoa is an efficient procedure to spread qualified semen samples for reproductive goals in commercial flocks, but the freeze-thawing process reduces the quality and fertility potential of post-thawed sperm cells. This study was aimed to investigate the effect of the mitochondria-targeted antioxidant MitoQ on rooster sperm quality and fertility potential preservation during freeze-thawing process. Semen samples were collected and diluted in the Lake medium, assigned into five equal aliquots, supplemented with 0, 1, 10, 100 and 1000 nM MitoQ, and cryopreserved in liquid nitrogen. After thawing, sperm motility, membrane functionality, abnormal morphology, mitochondria active potential, acrosome integrity, viability, apoptosis status, lipid peroxidation, DNA fragmentation, ROS concentration and fertility potential were evaluated. According to the results, freezing extender supplementation with 10 and 100 nM MitoQ presented higher (P ≤ 0.05) total motility, progressive motility, average path velocity, membrane functionality, mitochondria active potential, acrosome integrity and viability compared to the other groups. On the other hand, lipid peroxidation, apoptosis rate, DNA fragmentation and ROS concentration were lower (P ≤ 0.05) in groups received 10 and 100 nM MitoQ compared to other groups. Moreover, fertility rate was higher in groups received 10 and 100 nM MitoQ compared to control group. Therefore, MitoQ is able to preserve quality parameters and fertility potential of post-thawed spermatozoa in rooster and it could be an effective additive for supplementation of rooster's semen cryopreservation medium during reproductive programs.

Evaluation of Post-Thaw Swim-Up Selection Combined With Glutathione Addition for Improvement of Sperm Chromatin Maturity in Normozoospermic Samples.

Cryopreservation of human semen alters the spermatozoal structure, resulting in a reduction in sperm function parameters. Various antioxidants may be able to slow or prevent this type of injury. Glutathione (GSH) has numerous antioxidant properties; supplementing the semen with GSH before freezing may assist in the restoration of post-thaw sperm functionality. To investigate the effect of adding 5mM of glutathione before freezing on human sperm cryosurvival. Semen samples were collected from 30 patients (22 normozoospermic and 8 asthenozoospermic) after 3-5 days of sexual abstinence. Following liquefaction, macro- and microscopic examinations were performed. The samples were then divided into two equal aliquots: the first aliquot received 5 mM of glutathione before freezing, and the second aliquot was considered the control (without glutathione). The samples were frozen using the rapid cryopreservation method and then preserved for 7-10 days in a liquid nitrogen tank before being thawed. Thawed samples from each group were examined microscopically according to the WHO 2010guidelines. Aniline blue was used to assess the maturity of the DNA. Then, the direct-swim-up technique was applied to thawed samples from both groups to select the best sperm quality. The cryopreservation process negatively impacts all sperm characteristics in both groups. Sperm DNA integrity decreased. Glutathione addition before freezing decreased sperm chromatin immaturity (SCI) percent compared to the control (22.98 ± 0.83, 20.79 ± 0.56). The post-thaw performance of the swim-up technique resulted in a select sperm population with high progressive motility (p =0.04) andan improvement in DNA integrity in the treated group versus the control group after thawing. The DNA integrityin normozoospermic samples was improved by adding 5 mM glutathione before freezing. Performing the swim-up technique after thawing had no effect on sperm quality in asthenozoospermic patients.

Epsilon poly-lysine in buffalo semen extender: A step towards reducing the development of antibiotic resistance.

The use of antibiotics in semen extenders can contribute to the development of antibiotic resistance. The objective of the study was to evaluate epsilon-polylysine (Ɛ-PL) as a substitute for antibiotics in the buffalo semen extender. For this, 20 semen ejaculates were collected from four Murrah buffalo bulls. Each ejaculate was divided into three equal aliquots and extended into an egg yolk-based semen extender containing either antibiotics (strepto-penicillin) or different concentrations of Ɛ-PL (0.64 and 1.28 g/L) to make the final concentration 80 million sperm/mL and cryopreserved as per the standard procedure. The antibiogram sensitivity test confirmed that Ɛ-PL is an effective antimicrobial against microbes present in buffalo semen ejaculates. Furthermore, the addition of Ɛ-PL in the semen extender significantly reduces the colony forming unit (CFU)/mL in cryopreserved semen equivalent to strepto-penicillin. The sperm motility and kinematic parameters assessed by a computer-assisted sperm analyser showed that Ɛ-PL did not inhibit either sperm motility not kinematic parameters of cryopreserved sperm. The flow-cytometric evaluation of frozen-thawed sperm revealed interesting results. The extender supplemented with Ɛ-PL protected sperm acrosome and mitochondrial membrane potential greater than the extender supplemented with strepto-penicillin. Further, Ɛ-PL reduced significantly the production of superoxide anions from mitochondria during the cryopreservation process. In this way, Ɛ-PL may be a suitable alternative to antibiotics in semen extenders. In conclusion, Ɛ-PL at a concentration of 0.64 g/L acts as an effective antimicrobial as well as antioxidant in semen extender for cryopreservation of buffalo sperm.

Performance of CellDetect for detection of bladder cancer: Comparison with urine cytology and UroVysion.

To compare the performance of CellDetect, a new biomarker with urine cytology and UroVysiontechnology for bladder cancer detection. We performed an IRB approved prospective, blinded single center study in patients on routine surveillance for nonmuscle invasive bladder cancer and those scheduled for transurethral resection of bladder tumor or radical cystectomy. Patients with bladder catheters, neobladder, ileal conduit, urinary stones, or those with upper tract carcinoma were excluded from the study. Voided urine sample was collected from the participants and each sample was divided into three equal aliquots (CellDetect, Urine cytology and Urovysion). Pathology of the operative specimen was considered the gold standard to which the three markers were compared. The study group included 93 patients with median age was 68 years (range: 34-92 years) with male to female ratio of 12:1. Pathologic evaluation revealed malignancy in 43 cases (46%) of whom 81% had previous history of urothelial bladder cancer. Among all studied markers CellDetect exhibited the best performance followed by urine cytology and U-FISH with diagnostic odds ratio of 4.33, 3.85, and 2.5 respectively. The overall sensitivity, specificity, negative predictive value, and positive predictive value for this test were 84%, 80%, 88%, and 74% respectively. The advantage of this new biomarker was observed both in high grade and low-grade cases. This study demonstrates the advantage of CellDetect as a urine-based assay to detect urothelial bladder cancer over urine cytology and U-FISH test. The high performance was maintained across all cancer grades and stages without compromising the assay specificity. Additional studies are required to test if it can be a noninvasive alternative to cystoscopy.

Antioxidant effects of zinc-oxide nanoparticles on post-thaw quality and in vivo fertility of Beetal buck spermatozoa

Metallic nanoparticles are generally considered potential cryoprotectants during semen cryopreservation owing to their ability to improve the spermatozoa quality following cryopreservation when added in cryo-diluents. The present study aimed at investigating the effects of incorporating zinc oxide nanoparticles (ZnO-NPs) in the cryo-diluent on in vitro post-thaw sperm quality attributes, oxidant-antioxidant profile, and in vivo fertility rate in Beetal buck. After characterization, ZnO-NPs were added to the Tris-citric acid-fructose yolk extender. Semen samples, collected from five breeding bucks (5 ejaculates per buck) using an artificial vagina, were pooled. The pooled semen samples, distributed into five equal aliquots, were diluted in the Tris-citric acid cryo-diluent containing five different concentrations of ZnO-NPs (0.5 mg/ml, 1.0 mg/ml, 1.5 mg/ml, 2.0 mg/ml, and 0.0 mg/ml called control group), and were cryopreserved for 24 h. At post-thaw, various in vitro sperm quality parameters, and antioxidant enzyme profiles like superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), and reduced glutathione (GSH) levels were determined. For the in vivo fertility experiment, breeding does (n = 152), aging from 1.5 to 4 years, were artificially inseminated with spermatozoa with intact acrosome containing the same five concentrations of ZnO-NPs. Results demonstrated that the 1.0 mg/ml ZnO-NPs (crystallite size of 19.73 nm) improved (p < 0.01) the activities of antioxidant enzymes (SOD, CAT, and POD) with concomitantly decreased levels (p < 0.001) of lipid peroxidation (LPO) and reactive oxygen species (ROS) when compared with the other groups. Total spermatozoa motility, rapid velocity, and average path velocity were higher (p < 0.05) with the 1.0 mg/ml ZnO-NPs when compared with the control group. Progressive motility and straight-line velocity were higher (p < 0.05) with the 0.5, 1.0, and 1.5 mg/ml of ZnO-NPs addition compared with the 2.0 mg/ml and control groups. The sperm kinematics parameters; the amplitude of the lateral head displacement, the beat cross frequency, the straightness, and the linearity were not influenced by the ZnO-NPs inclusion. Supra-vital plasma membrane integrity was higher (p < 0.001) with the 0.5 mg/ml and 1.0 mg/ml of ZnO-NPs added groups when compared with either 2.0 mg/ml ZnO-NPs or the control group. The viable sperm with intact acrosome was higher (p < 0.05) in the 1.0 mg/ml ZnO-NPs group compared with the other groups. Low supplementation rates improved (p < 0.05) the DNA integrity when compared with the higher dose (2.0 mg/ml ZnO-NPs). The pregnancy per AI was higher (59.3 %; P < 0.05) in the does inseminated with semen added with the 0.5 mg/ml ZnO-NPs followed by 1.0 mg/ml (58.8 %), 1.5 mg/ml (58.6 %), and 2.0 mg/ml (46.6 %) when compared with the control group (33.3 %). We concluded that the 1.0 mg/ml ZnO-NPs added in the citric acid-fructose yolk extender improved the post-thaw quality of Beetal buck spermatozoa in terms of elevated activities of endogenous antioxidant enzymes, improved structural and functional integrity along with higher fertility.