Storage Time Points Research Articles

Herbal yeast product, Equi-Strath®, alters the antioxidant status of stallion semen

Effects of a plasmolysed yeast product enriched with herbs, malt, honey and orange syrup on semen characteristics and oxidative status in stallions were evaluated. Twenty stallions (mean age ± standard deviation = 9.5 ± 4.5 years) were randomly divided into a treatment group (n = 10) receiving 0.06 mL/kg bodyweight of plasmolysed herbal yeast, and a control group (n = 10) receiving the same amount of placebo daily in the feed for 10 weeks. Ejaculates were collected weekly from all stallions starting at Week 0. Volume, sperm concentration, motility, and velocity were evaluated immediately, 24 and 48 h after cooled storage at 5 °C. At the two storage time points, membrane lipid peroxidation was determined using the BODIPY-C11. Additionally, blood samples were collected at Weeks 0, 1, 5 and 9, and analysed for antioxidant status, consisting of superoxide dismutase, cholesterol, thiobarbituric acid reactive substances, and non-esterified fatty acids. Due to the nature of the data, the Mann-Whitney U test was applied as preliminary analysis. The BODIPY-C11 in the semen was less at 24 h and greater at 48 h after collections in Week 1 to 3 (P < 0.01) and Week 1 to 10 (P < 0.05) compared with Week 0 in the treatment compared to control group. There were no significant differences between groups for all values for other seminal and blood variables evaluated. In conclusion, feed supplementation with plasmolysed herbal yeast temporarily improved the antioxidant status of stallion semen, which might be of benefit for preservation of cooled semen.

Effect of Storage Time of a Ceramic Primer on Microshear Bond Strength to Zirconia

Objective: This study assessed the effect of shelf life of a ceramic primer containing 10-methacryloyloxydecyl dihydrogen phosphate (MDP) monomer on microshear bond strength of zirconia ceramic to composite resin.&#x0D; Materials and Methods: Sixty-four sintered zirconia samples (1.5×5×7 mm) were pretreated with Clearfil Ceramic Primer (CCP) at baseline and after one, two and six months of storage at 6°C. Composite cylinders were fabricated using Tygon tubes (1 mm height, 0.7 mm diameter) and placed on treated zirconia blocks, light-cured and subjected to microshear bond strength test. Mode of failure was determined under a stereomicroscope. Fourier-transform infrared spectroscopy (FTIR) was performed at each storage time point. Data were analyzed using one-way ANOVA and Tukey’s test. &#x0D; Results: Significant differences were noted in microshear bond strength after six months of storage compared to baseline (P&lt;0.05). Significant differences were noted in FTIR spectra at the four time points (P&lt;0.05).&#x0D; Conclusions: The effectiveness of one-component ceramic primer in bonding to zirconia was significantly affected by the storage of this material in a time-dependent manner. Visible new peaks and changes in FTIR spectra over time indicated alterations in the composition of ceramic primer affecting its shelf life.

Micro-quantity straw as a carrier for cryopreservation of oligozoospermic semen samples: Effects of storage times and cryoprotectant

Application of an appropriate freezing carrier is crucial for improving post-thaw recovery of oligozoospermic samples. In this study, our purpose is developing a user-friendly, easy handling and close micro-quantity (MQ) straw along with different freezing media, for cryopreservation of oligozoospermic samples. Twenty oligozoospermic semen samples were collected and mixed with glycerol egg yolk citrate (GEYC) or Spermfreeze® (SPF) medium. The mixture was loaded into MQ straws, sealed and stored in liquid nitrogen (LN) vapor. After freezing, the straws were transferred into cryotube and plunged into LN. Post-thawed sperm parameters including motion characteristics, viability, membrane and DNA integrity were evaluated one and three months after cryopreservation. The post-thawed sperm parameters were significantly reduced in GEYC and SPF medium compared to fresh samples. No statistically significant differences were seen in sperm characteristics between the two storage times (i.e. month 1 vs. month 3). Furthermore, GEYC medium yielded higher motility, viability and membrane integrity compared to SPF at both storage time-points. Sperm DNA integrity was also improved in GEYC group compared to SPF after 1 month of storage. The findings of our study showed that application of MQ straw along with GEYC, as the cryoprotectant, was beneficial for cryopreservation of low count semen samples.

Intradonor reproducibility and changes in hemolytic variables during red blood cell storage: results of recall phase of the REDS-III RBC-Omics study.

Genetic determinants may underlie the susceptibility of red blood cells (RBCs) to hemolyze in vivo and during routine storage. This study characterized the reproducibility and dynamics of in vitro hemolysis variables from a subset of the 13,403 blood donors enrolled in the RBC-Omics study. RBC-Omics donors with either low or high hemolysis results on 4°C-stored leukoreduced (LR)-RBC samples from enrollment donations stored for 39 to 42 days were recalled 2 to 12 months later to donate LR-RBCs. Samples of stored LR-RBCs from the unit and from transfer bags were evaluated for spontaneous and stress-induced hemolysis at selected storage time points. Intradonor reproducibility of hemolysis variables was evaluated in transfer bags over two donations. Hemolysis data at serial storage time points were generated on LR-RBCs from parent bags and analyzed by site, sex, race/ethnicity, and donation frequency. A total of 664 donors were successfully recalled. Analysis of intradonor reproducibility revealed that osmotic and oxidative hemolysis demonstrated good and moderate reproducibility (Pearson's r = 0.85 and r = 0.53, respectively), while spontaneous hemolysis reproducibility was poor (r = 0.40). Longitudinal hemolysis in parent bags showed large increases over time in spontaneous (508.6%) and oxidative hemolysis (399.8%) and smaller increases in osmotic (9.4%) and mechanical fragility (3.4%; all p < 0.0001). Spontaneous hemolysis is poorly reproducible in donors over time and may depend on site processing methods, while oxidative and osmotic hemolysis were reproducible in donors and hence could reflect consistent heritable phenotypes attributable to genetic traits. Spontaneous and oxidative hemolysis increased over time of storage, whereas osmotic and mechanical hemolysis remained relatively stable.

Concentrated emulsions as novel fat replacers in reduced-fat and low-fat Cheddar cheeses. Part 2. Large amplitude oscillatory shear behavior

Lower-fat cheeses with desirable texture can be difficult to produce. The objective of this study was to characterize the effects of concentrated emulsions as a fat replacer on the viscoelastic properties of reduced-fat (15% fat) and low-fat (6% fat) Cheddar cheeses. Concentrated emulsions were prepared by adding a fish gelatin–gum arabic mixture at pH 5.0 and 3.6 to olive oil at a water:oil ratio of 30:70 (w/w). Cheddar cheeses containing emulsions were made by adding the emulsions to skim milk after adding starter culture and before renneting and subsequent cheesemaking. Two control cheeses at each fat level were produced using either olive oil or cream. Cheeses containing emulsions showed greater nonlinearity under large amplitude oscillatory shear, and smaller critical stress and moduli values compared with controls at all storage timepoints. These results were ascribed to greater casein network discontinuity and more open spaces created by aggregated emulsion droplets.

α-Synuclein concentration increases over time in plasma supernatant of single donor platelets.

In platelets, α-synuclein is important in calcium-dependent granule release. Notably, cells release α-synuclein in setting of cell damage or death. Therefore, we investigated α-synuclein levels in plasma of single donor platelet (SDP) units during storage. Aliquots were obtained from same SDP units for 7days from day of donation. Additionally, randomly sampled SDP units at same storage time points were also assayed by enzyme-linked immunosorbent assay. α-Synuclein in SDP plasma increased continuously over time at each assayed time point. Significant increases were measured on day 3 (11.7±9.6ng/mL, P=0.025), day 5 (15.3±5.9ng/mL, P=0.002), and highest on day 7 (23.7±5.6ng/mL, P<0.0001) compared to day 0 (1.1±0.8ng/mL). Similar significant results were obtained in randomly sampled SDP units at same corresponding time points. Flow cytometry showed that platelets had strong expression of α-synuclein and lacked expression of other synucleins. Increases of α-synuclein during SDP storage is a steady and continuous process that increases with time. Our findings indicate that α-synuclein may represent a biomarker of platelet biological state during storage. Further research will be needed to determine how α-synuclein increases correlate with platelets' function.

Time and temperature dependent analytical stability of dry-collected Evalyn HPV self-sampling brush for cervical cancer screening

As a new initiative, HPV self-sampling to non-attenders using the dry Evalyn self-sampling brush is offered in the Capital Region of Denmark. The use of a dry brush is largely uncharted territory in terms of analytical stability. In this study we aim to provide evidence on the analytical quality of dry HPV self-sampling brushes as a function of time and temperature.We assessed the analytical stability of dry stored Evalyn brushes at three different temperatures, (4 °C, room temperature, 30 °C) and five different storage time points; T = 0 (baseline), 2, 4, 8, 16, and 32 weeks prior to HPV analysis using the BD Onclarity HPV assay.Mean Ct value of the Onclarity internal control was used as comparator of cellularity across time and temperatures, with no or only borderline statistical differences observed. HPV detection was stable throughout the five time points. In addition, analytically amplifiable DNA copy numbers and DNA fragmentation was assessed using the Agena iPLEX Exome QC assay, with no or only borderline statistical differences observed.In conclusion, the Evalyn brush is analytically stable with respect to human genomic material and HPV detection for up to 32 weeks at temperatures ranging from 4 °C to 30 °C.

Developmental capacity of Ascaridia galli eggs is preserved after anaerobic storage in faeces

The reliability of the results of in vitro studies such as detection of anthelmintic resistance often depends on the ability of the parasite eggs to develop under laboratory conditions. The aim of this study was to assess the embryonation capability of the chicken roundworm Ascaridia galli eggs after storage under different conditions. Two storage media for parasite eggs were used; faeces or water. Eggs in petri dishes (90 dishes in total) containing faces or water media were first exposed either to aerobic or anaerobic conditions at different temperatures (4 °C /+O2, 4 °C /−O2, 25 °C/−O2) for a maximum of 72 days. Every second week, materials from petri dishes in triplicates were recovered and incubated aerobically for two weeks at 25 °C. After the incubation, 200–300 eggs from each petri shish (sampling unit) were counted and the number of embryonated eggs was determined. Data was analyzed in R (version 3.4.3) A logistic regression model with the probability of an egg to embryonate as dependent variable and conditions, storage medium and time points as fixed effects with quasibinomial distribution was run. Least-square means were calculated and pairwise comparisons were made with the fixed effect factors (condition, storage medium and time point). Eggs in faeces had a significantly (p ˂ 0.05) higher embryonation than those in water, irrespective of storage conditions. At 4 °C embryonation tended to decline over time under aerobic conditions irrespective of the storage medium, whereas it remained constant following storage at anaerobic conditions. In contrast, anaerobic storage at the 25 °C negatively affected egg development in both media, except for day 14 in faeces. Our major finding was that eggs in faeces under anaerobic conditions and at 4 °C retained the highest rate of development, with a minimum decline in their developmental capacity over time compared to cleaned eggs stored in water.

Effect of High Oxygen Partial Pressure on 4-Hydroxy-2-Nonenal Induced Myoglobin Oxidation, Oxidation-Reduction Potential, and Myoglobin Unfolding

ObjectivesHigh oxygen modified atmosphere packaging (MAP) is often employed by the meat industry to enhance the appearance of fresh meat. 4-hydroxy-2-nonenal (HNE) is an ɑ-, β-unsaturated aldehyde derived from the oxidation of Ɯ6–polyunsaturated fatty acids. Previous research has examined the effect of HNE on oxymyoglobin oxidation at atmospheric oxygen partial pressure (20%). However, limited knowledge is currently available on the effects of HNE on myoglobin redox stability under high oxygen conditions. Therefore, the objective of this study was to examine the effect of high partial pressure of oxygen on HNE induced myoglobin oxidation.Materials and MethodsOxymyoglobin (0.15 mM; pH 5.6) prepared via hydrosulfite-mediated reduction was mixed with HNE (1.05 mM; dissolved in water), whereas controls received an equal volume of water. Following mixing, both samples were assigned to either high oxygen partial pressure (78%) or atmospheric oxygen partial pressure packaging. The samples were incubated either in a coffin-style display case maintained at 2 ± 1°C (under continuous lighting) for 0, 2 or 6 d, or in an incubator for 0, 1, 2, or 3 h at 25°C. Metmyoglobin formation, oxidation–reduction potential, and myoglobin unfolding were evaluated on each of the storage time points. The experiments were replicated 3 times (n = 3) and the data were analyzed using the Mixed Procedure of SAS (SAS Inst. Inc., Cary, NC).ResultsMyoglobin oxidation, oxidation–reduction potential, and protein unfolding properties were not different (P > 0.05) between treatments on time 0 for samples incubated at 4 and 25°C. On d 2 and 6, the high oxygen partial pressure treatment resulted in increased metmyoglobin formation and protein unfolding (P < 0.05) than the atmospheric oxygen partial pressure samples. Moreover, atmospheric oxygen partial pressure treatment had greater oxidation–reduction potential than high oxygen partial pressure treatment. At 25°C, high oxygen partial pressure treatments exhibited greater myoglobin oxidation from 1 to 3 h of incubation (P < 0.05) than atmospheric conditions.ConclusionThe results suggest that high oxygen partial pressure accelerated HNE induced oxidation irrespective of temperature. Further research using molecular protein biomarkers may provide an understanding of the underlying mechanistic steps involved in the interaction of HNE with the myoglobin at higher oxygen partial pressure.

UV imaging of multiple unit pellet system (MUPS) tablets: A case study of acetylsalicylic acid stability.

The applicability of multispectral ultraviolet (UV) imaging in combination with multivariate image analysis was investigated to monitor API degradation within multiple unit pellet system (MUPS) tablets during storage. For this purpose, acetylsalicylic acid (ASA) layered pellets were coated with Eudragit® RL PO and compressed to MUPS tablets. These tablets were stored under four different conditions with different levels of relative humidity (0 and 75%) and temperature (21 and 40°C) and analysed at seven storage time points (0, 15, 40, 140, 165, 265, and 330d). The UV imaging results for estimation of the salicylic acid (SA) concentration as degradation product of ASA in the tablets were compared to the SA concentration measured by high performance liquid chromatography with a partial least squares regression resulting in an RMSEP of 4.86% and an R2 of 0.9812. The estimation of the SA concentration based on mean UV reflectance spectra was possible even through the coating of the API pellets and at low concentration levels. In addition, the distribution of the SA concentration on the tablet surfaces for different storage time periods was visualized. UV imaging as fast and non-destructive method appears to offer significant potential for monitoring of API degradation during stability studies.

Microbial challenge test of a novel epoprostenol sodium formulation.

AimThe aim of the current study was to present a comprehensive display of antimicrobial activity of a novel epoprostenol sodium formulation with respect to seven different microorganisms, two levels of inoculation (102–103 colony forming units [CFU]/mL and 105–106 CFU/mL), two diluents (sterile water for injection [SWI] and sterile saline [sodium chloride 0.9%] for injection [SSI]), two concentrations (3,000 ng/mL and 15,000 ng/mL), and seven different storage time points at two temperatures (up to 10 days at 2°C–8°C and 20°C–25°C).Materials and methodsAntimicrobial activity was evaluated for, 1) solutions at 3,000 ng/mL inoculated with 102–103 CFU/mL and 105–106 CFU/mL; and 2) solutions at 15,000 ng/mL inoculated with 102–103 CFU/mL and 105–106 CFU/mL. All solutions were stored for up to 10 days at 2°C–8°C and 20°C–25°C. Solutions were prepared by reconstitution and further dilution of an epoprostenol sodium formulation using SWI or SSI. Antimicrobial activity was measured after inoculation with seven species of bacteria, yeast, and mold.ResultsFor all solutions, after 10 days, no microbial growth with respect to initial inoculum was observed, with the exception of a few early time points when using SWI as diluent. Some microorganisms died off completely, whereas others remained stable overall or returned to initial levels. Prior to decreasing, some microorganisms displayed a slight initial increase, presumed to be caused by breakup of clusters. Storage temperature had a negligible influence on the results, whereas choice of diluent (SSI or SWI) impacted growth kinetics in that SSI had a greater antimicrobial effect than SWI.ConclusionUpon reconstitution and further dilution of the novel epoprostenol formulation to concentrations of 3,000 ng/mL and 15,000 ng/mL with SWI or SSI, the resulting solutions did not support growth of the tested microorganisms when stored at 2°C–8°C or 20°C–25°C for up to 10 days.

Factors affecting genotyping success in giant panda fecal samples.

Fecal samples play an important role in giant panda conservation studies. Optimal preservation conditions and choice of microsatellites for giant panda fecal samples have not been established. In this study, we evaluated the effect of four factors (namely, storage type (ethanol (EtOH), EtOH −20 °C, 2-step storage medium, DMSO/EDTA/Tris/salt buffer (DETs) and frozen at −20 °C), storage time (one, three and six months), fragment length, and repeat motif of microsatellite loci) on the success rate of microsatellite amplification, allelic dropout (ADO) and false allele (FA) rates from giant panda fecal samples. Amplification success and ADO rates differed between the storage types. Freezing was inferior to the other four storage methods based on the lowest average amplification success and the highest ADO rates (P < 0.05). The highest microsatellite amplification success was obtained from either EtOH or the 2-step storage medium at three storage time points. Storage time had a negative effect on the average amplification of microsatellites and samples stored in EtOH and the 2-step storage medium were more stable than the other three storage types. We only detected the effect of repeat motif on ADO and FA rates. The lower ADO and FA rates were obtained from tri- and tetra-nucleotide loci. We suggest that freezing should not be used for giant panda fecal preservation in microsatellite studies, and EtOH and the 2-step storage medium should be chosen on priority for long-term storage. We recommend candidate microsatellite loci with longer repeat motif to ensure greater genotyping success for giant panda fecal studies.

Red blood cell membrane water permeability increases with length of ex vivo storage

Water transport across the red blood cell (RBC) membrane is an essential cell function that needs to be preserved during ex vivo storage. Progressive biochemical depletion during storage can result in significant conformational and compositional changes to the membrane. Characterizing the changes to RBC water permeability can help in evaluating the quality of stored blood products and aid in the development of improved methods for the cryopreservation of red blood cells. This study aimed to characterize the water permeability (Lp), osmotically inactive fraction (b), and Arrhenius activation energy (Ea) at defined storage time-points throughout storage and to correlate the observed results with other in vitro RBC quality parameters. RBCs were collected from age- and sex-matched blood donors. A stopped flow spectrophotometer was used to determine Lp and b by monitoring changes in hemoglobin autofluorescence when RBCs were exposed to anisotonic solutions. Experimental values of Lp were characterized at three different temperatures (4, 20 and 37 °C) to determine the Ea. Results showed that Lp, b, and Ea of stored RBCs significantly increase by day 21 of storage. Degradation of the RBC membrane with length of storage was seen as an increase in hemolysis and supernatant potassium, and a decrease in deformability, mean corpuscular hemoglobin concentration and supernatant sodium. RBC osmotic characteristics were shown to change with storage and correlate with changes in RBC membrane quality metrics. Monitoring water parameters is a predictor of membrane damage and loss of membrane integrity in ex vivo stored RBCs.

Paving the Way for Dose Banding of Chemotherapy: An Analytical Approach.

Background: In an interdepartmental cooperation, we investigated the feasibility and benefits of implementing dose banding of chemotherapy at our medical center. Based on this concept, chemotherapy doses are clustered into bands of similar dosage levels, thereby allowing the preproduction of frequently used standard doses of drugs, with sufficient physicochemical stability. Although established practice in the United Kingdom, there is little published evidence of its introduction elsewhere. Methods: We performed an analysis of local prescribing practice (22,310 chemotherapies) and identified gemcitabine, 5-fluorouracil, and carboplatin, among various others, as cytotoxic drugs suitable for dose banding. Results: First, we determined the physicochemical stability of the selected chemotherapy drugs during 12-weeks' storage by performing pH analysis and visual examination for color change or particles. No relevant changes were identified. Gemcitabine was selected for quantitative high-performance liquid chromatography analysis and we were able to show that ≥95% remained after 12 weeks' storage, in accordance with international guidelines. To simulate a worst case scenario, we performed microbiological stability testing of simulated cytotoxic compounding by replacing the cytotoxic drug with liquid media. Samples were incubated over defined storage time points (3, 6, and 12 weeks) and evaluated using the direct inoculation method. For the container integrity test, we deposited the samples into highly contaminated broth for 1 hour. Microbiological stability was demonstrated in both tests for the full storage period. Conclusions: Our data show that 12-weeks' storage of selected cytotoxic products is feasible from a microbiological perspective. Sterility of prepared products was maintained under extreme storage conditions. Gemcitabine content was in accordance with international guidelines after 12-weeks' storage. These results support the introduction of dose-banded gemcitabine products with the predicted advantages of optimized pharmacy workflow and reduced patient waiting times. We highlight the need for further research and consensus on the performance of purity analyses in dose-banded drug products.

Comparison of established and novel methods for the detection and enumeration of microparticles in canine stored erythrocyte concentrates for transfusion.

Microparticles (MP) are submicron, phosphatidylserine (PS)-bearing lipid vesicles with physiologic and pathologic roles in coagulation and inflammation. Microparticles accumulate in packed RBC (pRBC) stored for transfusion, potentially increasing recipient morbidity. Historically, canine MP have been detected with the PS label annexin V in supernatant samples. Other detection methods are available but have not been evaluated in dogs. The purpose of the study was to detect and enumerate MP in canine pRBC using annexin V, lactadherin, and bio-maleimide to compare label performance and assess microparticle accumulation under standard storage conditions. Microparticles (0.5-1.0 μm) in canine dog erythrocyte antigen 1.1 positive, nonleukoreduced pRBC were labeled with FITC-annexin V, FITC-lactadherin, and the fluorescent dye bio-maleimide, and were counted using flow cytometry at 3 time points (days 7, 21, and 35) of storage. Unprocessed pRBC, rather than supernatant, were used. Annexin V and bio-maleimide labeling produced comparable microparticle counts (P = .16), while lactadherin labeling resulted in higher microparticle counts than annexin V (P = .002) and bio-maleimide (P = .006), particularly on day 7. Bio-maleimide- and annexin V-based microparticle counts increased significantly from day 7 to 35 (P = .04), and increases from day 21 to 35 approached statistical significance (P = .05). Bio-maleimide- and annexin V-mediated microparticle counts were comparable in unprocessed canine pRBC using flow cytometry. Whether the increased microparticle counts with lactadherin were due to increased sensitivity for small, PS-bearing MP or due to labeling of membrane fragments and debris requires further investigation.

Importance of Donor Chondrocyte Viability for Osteochondral Allografts

Background: Osteochondral allograft (OCA) transplantation provides a biological treatment option for functional restoration of large articular cartilage defects in multiple joints. While successful outcomes after OCA transplantation have been linked to viable donor chondrocytes, the importance of donor cell viability has not been comprehensively validated. Purpose: To use a canine model to determine the importance of donor chondrocyte viability at the time of implantation with respect to functional success of femoral condylar OCAs based on radiographic, gross, cell viability, histologic, biochemical, and biomechanical outcome measures. Study Design: Controlled laboratory study. Methods: After approval was obtained from the institutional animal care and use committee, adult female dogs (N = 16) were implanted with 8-mm cylindrical OCAs from male dogs in the lateral and medial femoral condyles of 1 knee. OCAs were preserved for 28 or 60 days after procurement, and chondrocyte viability was quantified before implantation. Two different storage media, temperatures, and time points were used to obtain a spectrum of percentage chondrocyte viability at the time of implantation. A successful outcome was defined as an OCA that was associated with graft integration, maintenance of hyaline cartilage, lack of associated cartilage disorder, and lack of fibrillation, fissuring, or fibrous tissue infiltration of the allograft based on subjective radiographic, gross, and histologic assessments at 6 months after implantation. Results: Chondrocyte viability ranged from 23% to 99% at the time of implantation. All successful grafts had >70% chondrocyte viability at the time of implantation, and no graft with chondrocyte viability <70% was associated with a successful outcome. Live-dead stained sections and histologic findings with respect to cell morphological features suggested that successful grafts were consistently composed of viable chondrocytes in lacunae, while grafts that were not successful were composed of nonviable chondrocytes with infiltration of fibroblasts from the surrounding recipient tissues. In situ polymerase chain reaction (fluorescence in situ hybridization [FISH]) assays were performed in an attempt to distinguish donor (male) cells from recipient (female) cells. Unfortunately, this technique was exceptionally difficult to perform on intact articular cartilage sections, and consistent, repeatable data could not be obtained from this testing. However, the data did support histologic and live-dead data, which strongly suggested that successful grafts retained viable donor (male) chondrocytes and unsuccessful grafts degraded and were replaced by fibrous tissue populated with recipient (female) fibroblasts. Conclusion: Viable chondrocytes in OCAs at the time of transplantation are primarily responsible for maintenance of donor articular cartilage health in the long term. Clinical Relevance: Optimizing chondrocyte viability in all aspects of OCA transplantation—including procurement, processing, storage, transportation, and surgical implantation—needs to be a primary focus for OCA clinical use.

Importance Of Donor Chondrocyte Viability in Osteochondral Allografting

Objectives:Osteochondral allografting (OCA) provides a biologic treatment option for functional restoration of large articular cartilage defects in multiple joints. While successful outcomes after OCA have been linked to viable donor chondrocytes, the importance of donor cell viability has not been comprehensively validated. The purpose of this study was to use a canine model to determine the importance of donor chondrocyte viability at the time of implantation with respect to functional success of femoral condylar OCAs based on gross, cell viability, histologic, biochemical, and biomechanical outcome measures.Methods:With IACUC approval, adult female dogs (n = 16) were implanted with 8-mm cylindrical osteochondral allografts from male dogs in the lateral and medial femoral condyles of one knee. Osteochondral allografts were preserved for 28 or 60 days after procurement and chondrocyte viability was quantified prior to implantation. Two different storage media, temperatures and time points were used in order to obtain a spectrum of %chondrocyte viability at the time of implantation. A successful outcome was defined as an osteochondral allograft that was associated with graft integration, maintenance of hyaline cartilage, lack of associated cartilage disorder, and lack of fibrillation, fissuring, or fibrous tissue infiltration of the allograft based on subjective radiographic, arthroscopic, gross, and histologic assessments at 6 months after implantation.Results:Chondrocyte viability ranged from 23% to 99% at the time of implantation. All successful grafts had greater than 70% chondrocyte viability at the time of implantation and no graft with chondrocyte viability <70% was associated with a successful outcome. Live-dead stained sections and histologic findings with respect to cell morphology suggested that successful grafts were consistently comprised of viable chondrocytes in lacunae, while grafts that were not successful were comprised of non-viable chondrocytes with infiltration of fibroblasts from the surrounding recipient tissues. In situ PCR (FISH) assays were performed in an attempt to distinguish donor (male) cells from recipient (female) cells. Unfortunately, this technique was exceptionally difficult to perform on intact articular cartilage sections, and consistent, repeatable data could not be obtained from this testing. However, the data did support histologic and live-dead data, which strongly suggested that successful grafts retained viable donor (male) chondrocytes and unsuccessful grafts degraded and were replaced by fibrous tissue populated with recipient (female) fibroblasts.Conclusion:Viable chondrocytes in osteochondral allografts at the time of transplantation are primarily responsible for maintenance of donor articular cartilage health long term. Optimizing chondrocyte viability in all aspects of osteochondral allografting - including procurement, processing, storage, transportation, and surgical implantation - needs to be a primary focus for clinical use of OCA.

Effects of different anticoagulants, disposing time and processing methods on glucose concentration

Objective To explore the effects of different anticoagulants,disposing time,and processing methods on glucose concentration.Methods 120 samples of venous blood were collected from healthy volunteers with empty stomach.According to different anticoagulants in the vacuum tube and different methods of specimen processing,the samples were divided into six groups:group A (green tube),group B (gray tube),group C (yellow tube),group D (red tube),group E (green tube),and group F (gray tube).The specimens of the first four groups were placed at room temperature after centrifugation,the specimens of the last two were placed at room temperature but were not centrifuged.Blood glucose was detected at hours 0,2,4,6,and 8.Results Blood glucose levels differed significantly in groups A and E at different time points (P ＜0.05),the levels were gradually decreased by 13.88％ in group A and 40.69％in group E within 8 hours as prolongation of the disposing time; the volecity of decline was 1.73％/hour and 5.09％/hour,respectively.Blood glucose levels in groups B,C,and F did not differ significantly at different storage time points (P＞0.05).In group D,blood glucose concentration placed for 2 hours did not differ statistically as compared with that placed for 0 hour (P＞0.05),but the concentration for 4,6,and 8 hours differed statistically (P＜0.05); the concentration decreased 7.24％ within 8 hours and the volecity of decline was 0.91％ per hour.Conclusions Anticoagulants,disposing time,and processing methods have effects on blood glucose levels.Appropriate vacuum tubes are needed to collect blood and the samples should be centrifuged and detected as soon as possible. Key words: Glucose; Anticoagulant; Disposing time; Specimen processing

Quality Assessment of Longissimus and Semitendinosus Muscles from Beef Cattle Subjected to Non-penetrative and Penetrative Percussive Stunning Methods.

This study provides a comparative analysis of the effects of pre-slaughter penetrative and non-penetrative stunning and post-slaughter stunning on meat quality attributes in longissimus lumborum (LL) and semitendinosus (ST) muscles in heifers. Ten animals were assigned to each of four treatment groups: i) animals were subjected to conventional Halal slaughter (a clean incision through the structures at the front of the upper neck - the trachea, oesophagus, carotid arteries and jugular veins) and post-cut penetrating mechanical stun within 10 to 20 s of the neck cut (Unstunned; US); ii) high power non-penetrating mechanical stunning followed by the neck cut (HPNP); iii) low power non-penetrating mechanical stunning followed by the neck cut (LPNP); and iv) penetrative stunning using a captive bolt pistol followed by the neck cut (P). For each carcass, muscle samples were removed within 45 min of slaughter, portioned and analysed for pH, cooking loss, water holding capacity (WHC), tenderness (WBS), lipid oxidation (TBARS) and color, over a two week storage period. Stunning did not affect pH and cooking loss. Significant differences in water holding capacity, tenderness, lipid oxidation and color were present at different storage time points. HPNP stunning resulted in lower WHC and color values, particularly lightness (L*), higher TBARS values and peak force values compared with those stunned using LPNP, P and US. These adverse effects on quality were mostly encountered in the ST muscle. In conclusion, the meat quality achieved using P, LPNP and US treatments was comparable, and no treatment stood out as considerably better than another.

Retention of Structure and Function of the Cat Germinal Vesicle after Air-Drying and Storage at Suprazero Temperature

The study explored a novel approach for preserving the maternal genome without the entire oocyte by air-drying the cat germinal vesicle (GV) in the presence of the disaccharide trehalose. Specifically, we examined GV structure and function after desiccation, storage at 4 °C (up to 32 wk), and rehydration including the ability to resume meiosis after injection into a fresh, conspecific cytoplast. In experiment 1, DNA integrity was similar to fresh controls after 1 and 4 wk storage in the presence of trehalose, but was more fragmented at later time points (especially after 32 wk). Nuclear envelope integrity was sustained in >90% of oocytes stored for 0, 4, or 16 wk regardless of protective treatment. In experiment 2, compacted, air-dried GVs were stored for 2 or 4 wk, rehydrated, and injected into fresh cytoplasts. After culture for 24 h in vitro, up to 73% of oocytes reconstructed with desiccated GVs preserved in trehalose resumed meiosis compared to 30% of those dried in the absence of the disaccharide. At each storage time point, trehalose presence during air-drying was advantageous for resumption of meiosis, with >20% of oocytes completing nuclear maturation to metaphase II. This demonstrates a potential for preserving the female genome using the GV alone and for multiple weeks after desiccation. Trehalose enhanced the process by retaining the ability of a dried and rehydrated GV to resume communication with the surrounding cytoplasm of the recipient oocyte to permit reaching metaphase II and likely sustain subsequent embryo development.