Hemocytometer Counts Research Articles

Impact of microglia isolation and culture methodology on transcriptional profile and function.

Microglial isolation and culturing methods continue to be explored to maximize cellular yield, purity, responsiveness to stimulation and similarity to in vivo microglia. This study aims to evaluate five different microglia isolation methods-three variants of microglia isolation from neonatal mice and two variants of microglia isolation from adult mice-on transcriptional profile and response to HMGB1. Microglia from neonatal mice, age 0-3days (P0-P3) were isolated from mixed glial cultures (MGC). We included three variations of this protocol that differed by use of GM-CSF in culture (No GM-CSF or 500pg/mL GM-CSF), and days of culture in MGC before microglial separation (10 or 21). Protocols for studying microglia from adult mice age 6-8weeks included isolation by adherence properties followed by 7days of culture with 100ng/mL GM-CSF and 100ng/mL M-CSF (Vijaya et al. in Front Cell Neurosci 17:1082180, 2023), or acute isolation using CD11b beads (Bordt et al. in STAR Protoc 1:100035, 2020. https://doi.org/10.1016/j.xpro.2020.100035 ). Purity, yield, and RNA quality of the isolated microglia were assessed by flow cytometry, hemocytometer counting, and Bioanalyzer, respectively. Microglial responsiveness to an inflammatory stimulus, HMGB1, was evaluated by measuring TNFα, IL1β, and IFNβ concentration in supernatant by ELISA and assessing gene expression patterns using bulk mRNA sequencing. All five methods demonstrated greater than 90% purity. Microglia from all cultures increased transcription and secretion of TNFα, IL1β, and IFNβ in response to HMGB1. RNA sequencing showed a larger number of differentially expressed genes in response to HMGB1 treatment in microglia cultured from neonates than from adult mice, with sparse changes among the three MGC culturing conditions. Additionally, cultured microglia derived from adult and microglia derived from MGCs from neonates display transcriptional signatures corresponding to an earlier developmental stage. These findings suggest that while all methods provided high purity, the choice of protocol may significantly influence yield, RNA quality, baseline transcriptional profile and response to stimulation. This comparative study provides valuable insights to inform the choice of microglial isolation and culture method.

Novel Colorimetric and Light Scatter Methods to Identify and Manage Peritoneal Dialysis-Associated Peritonitis at the Point-of-Care

IntroductionPeritoneal dialysis-related peritonitis is a common cause of transfer to haemodialysis, patient morbidity and is a risk factor for mortality. Associated patient anxiety can deter selection of PD for renal replacement therapy. Diagnosis relies on hospital laboratory tests, however this might be achieved earlier if such information was available at the point-of-care, thereby significantly improving outcomes. The presence of culturable microbes and the concentration of leukocytes in effluent both aid peritonitis diagnosis, as specified in the International Society for Peritoneal Dialysis diagnostic guidelines. Here we report the development of two new methods providing such information in simple point-of-care tests. MethodsOne approach uses a tetrazolium-based chemical reporting system, primarily focused on detecting bacterial contamination and associated vancomycin-sensitivity. The second approach uses a novel forward light-scatter device (QuickCheck) to provide an instant quantitative cell count directly from PD patient effluent. ResultsThe tetrazolium approach detected and correctly distinguished lab isolates, taking 10h to provide non-quantitative results. We compared the technical performance of the light scatter leukocyte counting approach with spectrophotometry, haemocytometer counting and flow cytometry (Sysmex) using patient effluent samples. QuickCheck had high accuracy (94%) and was the most precise (coefficient of variation <4%), showing minimal bias, overall performing similarly to flow cytometry. ConclusionsThese complementary new approaches provide a simple means to obtain information to assist diagnosis at the point-of-care. The first provides antibiotic sensitivity following ten hours incubation, whereas the second optical approach (QuickCheck), provides instant accurate total leukocyte count.

Non-invasive and probeless rapid in-vitro monitoring and quantification of HUVECs counts based on FFT impedimetery

Introduction: The endothelial cells derived from the human vein cord (HUVECs) are used as in-vitro models for studying cellular and molecular pathophysiology, drug and hormones transport mechanisms, or pathways. In these studies, the proliferation and quantity of cells are important features that should be monitored and assessed regularly. So rapid, easy, noninvasive, and inexpensive methods are favorable for this purpose. Methods: In this work, a novel method based on fast Fourier transform square-wave voltammetry (FFTSWV) combined with a 3D printed electrochemical cell including two inserted platinum electrodes was developed for non-invasive and probeless rapid in-vitro monitoring and quantification of human umbilical vein endothelial cells (HUVECs). The electrochemical cell configuration, along with inverted microscope images, provided the capability of easy use, online in-vitro monitoring, and quantification of the cells during proliferation. Results: HUVECs were cultured and proliferated at defined experimental conditions, and standard cell counts in the initial range of 12 500 to 175 000 were prepared and calibrated by using a hemocytometer (Neubauer chamber) counting for electrochemical measurements. The optimum condition, for FFTSWV at a frequency of 100 Hz and 5 mV amplitude, were found to be a safe electrochemical measurement in the cell culture medium. In each run, the impedance or admittance measurement was measured in a 5 seconds time window. The total measurements were fulfilled at 5, 24, and 48 hours after the seeding of the cells, respectively. The recorded microscopic images before every electrochemical assay showed the conformity of morphology and objective counts of cells in every plate well. The proposed electrochemical method showed dynamic linearity in the range of 12 500-265 000 HUVECs 48 hours after the seeding of cells. Conclusion: The proposed electrochemical method can be used as a simple, fast, and noninvasive technique for tracing and monitoring of HUVECs population in in-vitro studies. This method is highly cheap in comparison with other traditional tools. The introduced configuration has the versatility to develop electrodes for the study of various cells and the application of other electrochemical designations.

Quantitative multiplex PCR in copies ml-1 linearly correlates with standard urine culture in colonies ml-1 for urinary tract infection (UTI) pathogens.

Standard urine culture (SUC) is the current standard method for confirmation of a urinary tract infection (UTI). SUC identifies microorganisms in urine samples and semi-quantifies these as colony-forming units (CFU) ml-1. In contrast, quantitative multiplex polymerase chain reaction (q-MPCR) is a culture-independent assay in which the microbes are quantified by targeting genomic sequences and reported as cells ml-1, calculated from copies ml-1. Using serial dilutions within the 104-105 cells ml-1 range, the usual reporting range of SUC, this study compared the quantification results based on SUC and q-MPCR for four uropathogens with the control hemocytometer counts. The results revealed a linear relationship and a 1:1 correlation between the q-MPCR and SUC results. Additional q-MPCR quantification of 36 uropathogenic non-fastidious and fastidious bacteria and yeast indicated a reproducible linear correlation in a 1:1 manner with the control counts over a range of cell densities (103-106). The results confirm that the quantifications by q-MPCR in cells ml-1 and by SUC in CFU ml-1 are comparable and answer to the lingering question of how the results of these two methods correlate. Moreover, q-MPCR provided accurate quantification of various microorganisms over wider cell density ranges without the time required for microbial growth.

Conidial mass production of entomopathogenic fungi and tolerance of their mass-produced conidia to UV-B radiation and heat

We investigated conidial mass production of eight isolates of six entomopathogenic fungi (EPF), Aphanocladium album (ARSEF 1329), Beauveria bassiana (ARSEF 252 and 3462), Lecanicillium aphanocladii (ARSEF 6433), Metarhizium anisopliae sensu lato (ARSEF 2341), Metarhizium pingshaense (ARSEF 1545), and Simplicillium lanosoniveum (ARSEF 6430 and 6651) on white or brown rice at four moisture conditions (75–100%). The tolerance of mass-produced conidia of the eight fungal isolates to UV-B radiation and heat (45 °C) were also evaluated. For each moisture content compared, a 20-g sample of rice in a polypropylene bag was inoculated with each fungal isolate in three replicates and incubated at 28 ± 1 °C for 14 days. Conidia were then harvested by washing the substrate, and conidial concentrations determined by haemocytometer counts. Conidial suspensions were inoculated on PDAY with 0.002% benomyl in Petri plates and exposed to 978 mW m−2 of Quaite-weighted UV-B for 2 h. Additionally, conidial suspensions were exposed to 45 °C for 3 h, and aliquots inoculated on PDAY with benomyl. The plates were incubated at 28 ± 1 °C, and germination was assessed at 400 × magnification after 48 h. Conidial production was generally higher on white rice than on brown rice for all fungal species, except for L. aphanocladii ARSEF 6433, regardless of moisture combinations. The 100% moisture condition provided higher conidial production for B. bassiana (ARSEF 252 and ARSEF 3462) and M. anisopliae (ARSEF 2341) isolates, while the addition of 10% peanut oil enhanced conidial yield for S. lanosoniveum isolate ARSEF 6430. B. bassiana ARSEF 3462 on white rice with 100% water yielded the highest conidial production (approximately 1.3 × 1010 conidia g−1 of substrate). Conidia produced on white rice with the different moisture conditions did not differ in tolerance to UV-B radiation or heat. However, high tolerance to UV-B radiation and heat was observed for B. bassiana, M. anisopliae, and A. album isolates. Heat-treated conidia of S. lanosoniveum and L. aphanocladii did not germinate.

Amoebicidal effect of Fluconazole and Verapamil together against trophozoites and cysts of Acanthamoeba castellanii

Acanthamoeba species are an important pathogen that causes Acanthamoeba keratitis, which causes a visual loss, and encephalitis, which lead to the central nervous system infection and death. The options available to successfully treat Acanthamoeba infections have limited success. New therapeutic approaches must thus be developed, and especially combination medication therapy may be a successful and an effective strategy. The aim of this study was to assess the combination efficacy of verapamil and fluconazole against Acanthamoeba trophozoites and cysts. The effects of drugs on growth inhibition against Acanthamoeba were tested using amoebicidal assays. The viability of Acanthamoeba was assessed using Trypan blue and hemocytometer counts. The effect of three different concentrations of fluconazole, verapamil and fluconazole + verapamil combination on growth inhibition against Acanthamoeba trophozoites and cysts was significant compared to the control (p

Potential of Fumagillin Against &lt;i&gt;Nosema&lt;/i&gt; SP., a Microsporidian Endoparasite of Honey Bee &lt;i&gt;Apis mellifera&lt;/i&gt; L

Nosema apis and Nosema ceranae are two kinds of microsporidian parasites that cause the recently identified illness known as nosemosis in European honey bees (Apis mellifera). 10 samples of adult honey bees from each infected colony of a single apiary were collected, located in the village of Handia, district of Prayagraj, India, from September 2022 to January 2023 to investigate the frequency of intestinal infections and the effectiveness of fumagillin antibiotic against nosemosis in A. mellifera with the use of monocular LED optical microscopy and a haemocytometer, the mean and median spore count of each composite sample was calculated. It was observed that 5% of the infected gut samples contained Nosema spores. The results show that 10% of colonies tested positive for Nosema, and fumagillin is effective against nosemosis.

A Rapid Assessment of Microalgal Concentration Using Turbidity Measurement for Shellfish Hatchery Seed Production

The goal of this study was to develop a rapid assessment of microalgal concentration using a turbidity meter to serve commercial shellfish hatcheries for feeding shellfish larvae and broodstock. We used four commonly used algal species, Tetraselmis suecica, Isochrysis galbana, Chaetoceros calcitrans, and Chaetoceros gracilis, generating serial dilutions of each and measuring cell concentrations and turbidity of each sample. We identified linear correlations between cell concentration and turbidity and established standard equations between cell concentration and turbidity for each species. We sampled and quantified algae from a shellfish hatchery using these equations and compared hemocytometer counts. No differences were found (P ≥ 0.174), indicating the accuracy of the equations. We expect that this method can provide a quick, accurate, easy method for shellfish hatcheries to quantify algal concentration and avoid either over- or underfeeding larvae and broodstock.

Validation of the iSperm for assessing rhinoceros Sperm

This study's objective was to determine the feasibility of using a portable computer-assisted sperm analyzer to standardize the evaluation of rhinoceros sperm motility and concentration. Assessments were performed opportunistically as part of other ongoing studies and included both fresh and cryopreserved samples from black (Diceros bicornis; n = 3), white (Ceratotherium simum; n = 7), and greater one-horned (Rhinoceros unicornis; n = 5) rhinoceroses. Accuracy of the iSperm Rhino 5 software in identifying sperm cells was evaluated through manual scoring of sperm tracks (n = 48 videos from 12 samples). The number of sperm identified by the observer and the software were significantly correlated (Pearson r = 0.9908, P < 0.0001). Moreover, the bias between the observer to iSperm values was small (Bland-Altman test; mean ratio = 0.97 observer/iSperm). A strong correlation (Pearson r = 0.9847, P < 0.0001) between hemocytometer counting and the iSperm for sperm concentration (n = 50) was also observed with a bias of just 1.03 (mean ratio hemocytometer to iSperm). Reliability analysis was performed by comparing ten consecutive evaluations on four different samples using intraclass correlation coefficients (ICC). In this study, the reliability (aka repeatability) of measuring the concentration or total motility via iSperm was deemed excellent (ICC > 0.96). Results of this work demonstrate that the iSperm offers a reliable and semi-automated alternative to manual evaluations for assessing sperm concentrations and motility in the rhinoceros. The ability to determine wildlife sperm concentrations rapidly and motility objectively while working under field conditions would be a great asset to conservation biologists in both zoological institutions and wildlife reserves.

Establishment of preliminary reference intervals and cytochemical staining of blood cells in big-bellied seahorses (Hippocampus abdominalis).

Despite their popularity, hematology reference intervals (RIs) have not been established in big-bellied seahorses (Hippocampus abdominalis). The objectives of this study were to establish hematologic RIs to compare values between sex in regard to cytochemical staining of blood cells. We also sought to compare white blood cell concentrations using the Natt and Herrick technique vs blood smear estimates. Forty-three healthy individuals from the Aquarium du Québec (22 females and 21 males) were included. Normal health status was confirmed by an unremarkable physical examination in five individuals and by necropsy of five other individuals, of which all were excluded from further analyses. Venipuncture was performed from the ventral coccygeal vein in the remaining 33 individuals without anesthesia using heparinized insulin syringes. A blood volume of 0.05 to 0.1ml was collected to prepare Wright Giemsa-stained blood smears and hematocrits immediately after venipuncture. Whole blood was stored in heparinized Eppendorf tubes to determine red and white blood cell concentrations using the Natt and Herrick technique with a hemocytometer in 10 individuals; these results were compared with blood smear estimates. Additional blood smears were stained with alkaline phosphatase substrate, periodic acid Schiff, and toluidine blue stains. The reference intervals included the packed cell volume (27.4-67.5%), thrombocyte count (19.5-197.7 × 109 /L), and white blood cell (WBC) count (2-54.8 × 109 /L), including neutrophils (1.1-21.3 × 109 /L), lymphocytes (2.7-45.5 × 109 /L), and monocytes (0-2.2 × 109 /L). The WBC hemocytometer counts showed no correlation with blood smear estimates (Spearman's rho=0.2). There was also no significant difference between the sexes. These preliminary reference intervals will help assess the health of seahorses.

Automated enumeration of Eimeria oocysts in feces for rapid coccidiosis monitoring

Coccidiosis represents a major driver in the economic performance of poultry operations, as coccidia control is expensive, and infections can result in increased feed conversion ratios, uneven growth rates, increased co-morbidities with pathogens such as Salmonella, and mortality within flocks. Shifts in broiler production to antibiotic-free strategies, increased attention on pre-harvest food safety, and growing incidence of anti-coccidial drug resistance has created a need for increased understanding of interventional efficacy and methods of coccidia control. Conventional methods to quantify coccidia oocysts in fecal samples involve manual microscopy processes that are time and labor intensive and subject to operator error, limiting their use as a diagnostic and monitoring tool in animal parasite control. To address the need for a high-throughput, robust, and reliable method to enumerate coccidia oocysts from poultry fecal samples, a novel diagnostic tool was developed. Utilizing the PIPER instrument and MagDrive technology, the diagnostic eliminates the requirement for extensive training and manual counting which currently limits the application of conventional microscopic methods of oocysts per gram (OPG) measurement. Automated microscopy to identify and count oocysts and report OPG simplifies analysis and removes potential sources of operator error. Morphometric analysis on identified oocysts allows for the oocyst counts to be separated into 3 size categories, which were shown to discriminate the 3 most common Eimeria species in commercial broilers, E. acervulina, E. tenella, and E. maxima. For 75% of the samples tested, the counts obtained by the PIPER and hemocytometer methods were within 2-fold of each other. Additionally, the PIPER method showed less variability than the hemocytometer counting method when OPG levels were below 100,000. By automated identification and counting of oocysts from 12 individual fecal samples in less than one hour, this tool could enable routine, noninvasive diagnostic monitoring of coccidia in poultry operations. This approach can generate large, uniform, and accurate data sets that create new opportunities for understanding the epidemiology and economics of coccidia infections and interventional efficacy.

An investigation on cauda storage of sperm in hamster epididymis

Abstract Immature testicular sperm of mammals acquire the potential to move in a forward direction during their journey through excurrent ducts, efferent ductules and the epididymis. The present study aimed to determine the sperm storage time using the hamster cauda epididymis. Ligation of the left epididymal tubule at the junction between the distal corpus and the proximal cauda was carried out to determine the storage time. The right epididymis was left unaltered as the control. On days 3, 12, 15, 24, 28, 32, and 40 after ligation of the cauda, experiments were carried out and repeated five times. Sperm total count and mortality were determined using the haemocytometer counting method and sperm viability was assessed with the live/dead viability kit. The morphology of cauda sperm was observed by preparing sperm smears from each animal. Data were analyzed using SPSS and all values were expressed as mean ± SEM. On day 40, the total number of cauda sperms was reduced remarkably. In the experimental groups and in the control, 3–6% of sperm motility was maintained until day 40. By day 3, the percentage of live sperm was 50% and by the 40th day, it was decreased up to 10%. In the control group, the live sperm percentage was 24% by the 40th day (). By day 32, 76% of the cauda spermatozoa appeared abnormal with head defects, mid piece and neck defects and multiple defects. Findings of the present study indicate that cauda sperm storage time is more than 40 days. Motility, viability and morphology of these spermatozoa were decreased remarkably during this storage time.

Antiamoebic Activity of Imidazothiazole Derivatives against Opportunistic Pathogen Acanthamoeba castellanii.

We examined the antiamoebic effect of several imidazothiazole derivatives on Acanthamoeba castellanii of the T4 genotype. Trypan blue exclusion assays and haemocytometer counting were used to determine the reduction in A. castellanii trophozoite proliferation, in response to treatment with these compounds. To determine the effects of these compounds on host cells, lactate dehydrogenase assay was performed using HeLa cell lines. Amoebicidal assays revealed that the tested compounds at concentrations of 50 µM significantly inhibited amoebae trophozoites compared to controls. Compounds 1m and 1zb showed the highest amoebicidal effects eradicating 70% and 67% of A. castellanii, respectively. The compounds blocked both the encystation and excystation process in A. castellanii. Compounds 1m and 1zb blocked 61% and 55%, respectively, of amoeba binding to human cells. Moreover, the compounds showed minimal cytotoxic effects against host cells and considerably reduced amoeba-mediated host cell death. Overall, our study revealed that compounds 1m and 1zb have excellent antiamoebic potential, and should be considered in the development of curative antiamoebic medications in future studies. Further work is critical to determine the translational value of these findings.

SHORT-TERM BIOLOGICAL VARIABILITY OF HEMATOLOGY PARAMETERS IN THE BEARDED DRAGON (POGONA VITTICEPS)

Determination of the biological variation of laboratory tests is essential for accurate interpretation during health assessments. Few studies investigate the biological variation of CBC variables in reptiles, and none of these involve squamates. Thus, we investigated the biological variation of hematology parameters in bearded dragons (Pogona vitticeps) to determine if conventional population-based reference intervals are appropriate in evaluating single laboratory samples. Blood was collected from 10 bearded dragons five separate times over 10 wk and placed in lithium heparin (LH) microtainers, and CBC were performed. WBC were evaluated via both a fresh, non-anticoagulated blood smear and a LH anticoagulated blood smear as well as two manual hemocytometer counts with blood stained with either Natt and Herrick's (NH) solution or Leukopet™ (LO) solution. The between-animal coefficient of variation (CVG), within-animal coefficient of variation (CVI), reference change value (RCV), and index of individuality were determined for PCV, total solids (TS), and WBC using all leukocyte quantification methods. The RVC for PCV, TS, and WBC in bearded dragons were 37%, 84%, and >100%, respectively. The calculated index of individuality values all fell between 0.6 and 1.4, suggesting that the use of judiciously applied population-based reference intervals in interpreting the WBC, PCV, and TS in bearded dragons is appropriate.

A Biochemistry Course Undergraduate Research Experience (CURE) on Crithidia fasciculata parasites

Crithidia fasciculata is a non‐pathogenic parasite related to human‐infective parasites including Trypanosoma bruceiwhich causes African Sleeping Sickness, T. cruziwhich causes Chagas’ disease, and Leishmania spp. which causes leishmaniasis. We developed a Course Undergraduate Research Experience (CURE) on Crithidiaparasitology for a 300‐level biochemistry laboratory. Optical density at 600nm was used to study parasite cell density compared to hemocytometer counts of cells. At 27⁰C the population doubled every four hours. Carbon‐13 NMR was used to study Crithidia fasciculatametabolism of 1‐13C‐glucose. NMR peaks for the fermentation products ethanol and succinate increased over time. Competition experiments demonstrated that sucrose competed with glucose as a carbon source, fructose metabolism was less efficient than glucose, and xylose did not compete. Students also designed their own projects on Crithidia, building a toolbox of interesting stressors and successful assays to study biochemical parasitology with Crithidiaparasites.

Adjustment of common carp sperm concentration prior to cryopreservation: Does it matter?

Absorbance and computer-assisted sperm analysis (CASA) was used to assess the sperm concentration of common carp (Cyprinus carpio) as an alternative to hemocytometer counting. Also the effect of different sperm concentrations on post-thaw sperm parameters and fertilizing capacity were tested. The main goal of the research was to find out whether the tested methods are suitable for fast measurement of sperm concentration and if the pre-setting of sperm concentration before cryopreservation offers any advantages over dilution of sperm to a given ratio. A linear relationship of sperm concentration measured with CASA (N = 12) as well as sperm absorbance assessed with a microplate reader with spectrophotometry module (N = 18) with sperm concentration counted in a Bürker-Türk type hemocytometer was established. For cryopreservation, 4 sperm concentrations were used (0.5; 1; 2; 4 × 109 spermatozoa per ml) and compared to dilution to a ratio of 1:9. A significant positive relationship was found between the absorbance and sperm concentration (p < 0.0001, r2 = 0.8289) as well as between immotile cell concentration measured with CASA and that counted in a hemocytometer (p < 0.0001, r2 = 0.8559). No significant main effect of sperm concentration was found on any post-thaw motility parameters measured by CASA with the exception of linearity (p = 0.0056). Sperm concentration had a significant main effect (p = 0.0156) on the fertilizing capacity of sperm samples diluted to various concentrations prior to cryopreservation. Only the highest concentration of 4 × 109 spermatozoa per ml (66 ± 6% fertilization) showed a significant difference (p = 0.0121) in comparison to the pre-set dilution ratio (49 ± 5% fertilization). The results show that both spectrophotometry and CASA are suitable for the assessment of common carp sperm concentration and that maximizing sperm concentration prior to cryopreservation results in higher fertilization.

Sperm production and quality in European eel (Anguilla anguilla) in relation to hormonal treatment.

Aquaculture production relies on controlled management of gametogenesis, especially in species where assisted reproduction is needed for obtaining gametes in captivity. The present study used human chorionic gonadotropin (hCG) treatments to induce and sustain spermatogenesis in European eel (Anguilla anguilla). The aim was to evaluate effects of strip-spawning timing (12 vs. 24hr) after weekly administration of hCG and the necessity of a primer dose (in addition to weekly hormonal treatment) prior to strip spawning (primer vs. no-primer) on sperm quality parameters. Sperm parameters included milt production (weight), density and sperm kinematics at Week 9, 11 and 13 after onset of treatment. Spermiation commenced in 11.5% of males in Week 5 and by Week 9, and all males produced milt. Male weight, milt production, sperm density and spermatocrit did not differ among hormonal treatments during the experimental period. Overall, male weight decreased from 106.3 to 93.0g, milt weight increased from 3.5 to 5.4g, sperm density counts decreased from 11.7×109 to 10.5×109 cells/ml, and spermatocrit decreased from 46.5% to 40.5%. Furthermore, spermatocrit was positively related to haemocytometer counts (R2 =.86, p<.001), providing a reliable indicator of sperm density. Differences in sperm kinematics were observed depending on strip-spawning timing after hormonal injection (12 vs. 24hr) but with no consistent pattern. These sperm quality parameters also did not consistently differ between the no-primer and primer treatments. Considering that each male may be stripped 4-5 times over the 2-3months spawning season, omitting the primer would reduce animal handling, material costs and labour intensity, while sustaining high-quality sperm production.

Hypertonic Dextrose Stimulates Chondrogenic Cells to Deposit Collagen and Proliferate.

Hypertonic dextrose (HD) injections (prolotherapy) for osteoarthritis are reported to reduce pain. Cartilage regeneration is hypothesized as a mechanism. This in vitro study identifies an HD concentration that stimulates chondrogenic cells to increase metabolic activity and assesses whether this concentration affects collagen deposition and proliferation. ATDC5 chondrogenic cells were cultured in normoglycemic DMEM/F12 medium, treated with concentrations of HD (4-400 mM), and assessed with PrestoBlue. Advanced light microscopy was used to conduct live imaging of collagen deposition through second harmonic generation microscopy (SHG) and proliferation via 2-photon excitation microscopy. Proliferation was additionally assessed with hemocytometer counts. A linear regression model found that, relative to the 4 mM baseline control, cells treated with 200 mM had a higher mean absorbance (P = 0.023) and cells treated with 250 mM were trending toward a higher mean absorbance (P = 0.076). Polynomial regression interpolated 240 mM as producing the highest average absorbance. Hemocytometer counts validated 250 mM as stimulating proliferation compared with the 4 mM control (P < 0.01). A concentration of 250 mM HD led to an increase in collagen deposition compared with that observed in control (P < 0.05). This HD concentration also led to increases in proliferation of ATDC5 cells relative to that of control (P < 0.001). A 250 mM HD solution appears to be associated with increased metabolic activity of chondrocytes, increased collagen deposition, and increased chondrocyte proliferation. These results support clinical prolotherapy research suggesting that intra-articular HD joint injections reduce knee pain. Further study of HD and cellular processes is warranted.

Development of methodology and reference intervals for the analysis of the free-ranging Atlantic horseshoe crab Limulus polyphemus hemolymph.

The Atlantic horseshoe crab, Limulus polyphemus, is valuable to fishing and biomedical industries, for education in public aquaria, and of ecological importance because the eggs provide a critical food source for migratory shorebirds. While hematologic and biochemical analyses are fundamental tools for health assessment, reports are limited for the four extant species of horseshoe crabs; Lpolyphemus (East Coast North America), Carcinoscorpius rotundicauda (mangrove horseshoe crab Southeast Asia), Tachypleus gigas, and Tachypleus tridentatus (Southeast and East Asia). This prospective study measured hemolymph values to determine RIs from free-ranging horseshoe crabs during the annual breeding aggregation in Delaware Bay, USA. Near-shore animals were briefly manually restrained for hemolymph collection from the cardiac sinus. Hematologic samples (25 males, 25 females) were collected for hemocytometer counts using the Natt and Herrick method. A second set of 50 animals (25 males, 25 females) was sampled for biochemistry measurands, including copper concentration by spectrophotometer, osmolality by vapor pressure osmometer, and protein electrophoresis. Total hemocyte counts varied widely (RI 6600-44300/µL). Electrolytes were consistent with osmoconformers living in seawater. There was a marked difference in sexes, with females showing much lower protein, glucose, and copper levels. Eleven females had multiple outliers, and all data were excluded from RI calculations. Further studies of this species outside of spawning season are needed. Expanding our knowledge of horseshoe crab health is important to decreasing morbidity and mortality in research applications and maximizing longevity and overall health in display settings.

Reproductive aspects of freshwater unionid mussel sperm: Seasonal dynamics, male-to male variability, and cell quantification

Freshwater unionid mussel diversity is decreasing because of species extirpation or extinction. While little can be done to recover lost species, there is an opportunity to develop techniques to save other species. This can be facilitated through gene banking and assisted reproduction. Unfortunately, limited information is available on mussel reproduction, especially relating to sperm quality. Objectives, therefore, were to quantify seasonal changes in sperm concentration and morphology for two unionid mussels, Ligumia subrostrata and Lampsilisstraminea, measure intraspecific heterogeneity for sperm morphometry, and develop an efficient method to quantify sperm concentration using a microspectrophotometer. There were no differences in sperm concentration when cells were extracted from the center or at a half centimeter on either side of the visceral mass, during the spawning season. There was a seasonal change in sperm concentration, such that concentration for L. subrostrata ranged from 1.1 × 109 to 19.60 × 109 cells/mL with there being the largest counts between 26 September to 7 November. L. straminea sperm concentration was greatest (20.0 × 109 cells/mL) on 13 September and subsequently decreased. Sperm were uniflagellated and SEM results for L. subrostrata and L. straminea showed mean head length and width (mid-spawning) were 3.38 ± 0.04 μm and 1.61 ± 0.01 μm and 3.37 ± 0.04 μm and 1.61 ± 0.01 μm, respectively. There were close (R2 ≥ 0.85) quadratic associations between hemocytometer counts and absorbance (300, 600, 700 nm). These results provide baseline information to further investigate sperm quality, fertilizing capacity, and cryopreservation for freshwater mussels.