Short-term Storage Conditions Research Articles

Investigation of optimal sperm storage conditions for short-term storage

Objective The quality of sperm cells is important role in the success rates of assisted reproduction technology (ART) treatments. The quality of the sperm cells shows variations depending on the temperature of short-term semen storage as well as the methods of semen preparation. Thus, this study aimed to investigate the sperm viability, motility and DNA fragmentation following different sperm preparation methods and short-term storage conditions, respectively. Materials and Methods A total of 25 semen samples were evaluated. In the first part of this study, different incubation temperatures were investigated in two groups, in such the first group involved the semen samples and the second group involved the sperm cells separated by density gradient centrifugation method, respectively. The samples in each group were incubated at 4°C, room temperature (21°C) and 37°C for 24 hours, respectively. The sperm cell qualities were evaluated by mobility analysis, DNA fragmentation by acridine orange staining and sperm cell viability by propidium iodide staining. Results and Discussion The analysis outcome demonstrated that the mobility, DNA fragmentation and viability of the sperm cells were statistically different when incubated at RT (21°C) in both groups. Furthermore, samples prepared by the density gradient centrifugation method were shown to have better quality. The optimum short-term storage temperature was detected to be the room temperature. Conclusion The conclusion of this investigation is crucial to assess storage conditions in ART clinics. This study provides essential data for short-term sperm storage and preparation methods to improve the success rates of ART clinics. Bangladesh Journal of Medical Science Vol. 23 No. 04 October’24 Page : 1137-1141

Effect of short-term sample storage and preparatory conditions on losses of 18 per- and polyfluoroalkyl substances (PFAS) to container materials

There is a lack of agreement on a suitable container material for per- and polyfluoroalkyl substances (PFAS) analysis, particularly at trace levels. In this study, the losses of 18 short- and long-chain (C4–C10) PFAS to commonly used labware materials (high-density polyethylene (HDPE), polypropylene (PP), polystyrene (PS), polypropylene co-polymer (PPCO), polyethylene terephthalate (PET), polytetrafluoroethylene (PTFE), and glass were investigated. The influence of sample storage and preparation conditions, i.e., storage time, solvent composition, storage temperatures (4 °C and 20 °C), and sample agitation techniques (shaking and centrifugation) on PFAS losses to the container materials were investigated. The results showed higher losses for most of the considered PFAS (up to 50.9%) in 100% aqueous solutions after storage for 7 days regardless of the storage temperature compared to those after 3 days. Overall, the order of losses to different materials varied for individual PFAS, with the highest losses of long-chain PFAS observed to PP and HDPE after 7-day storage at room temperature. The addition of methanol to aqueous PFAS solutions reduced the losses of long-chain PFAS to all tested materials. The use of sample centrifugation and shaking did not influence the extent of losses for most of the PFAS in 80:20 water:methanol (%, v/v) to container materials except for 8:2 fluorotelomer sulfonic acid (8:2 FTS), 9-chlorohexadecafluoro-3-oxanone-1-sulfonic acid (9Cl-PF3ONS), perfluorodecanoic acid (PFDA) and 4:2 fluorotelomer sulfonic acid (4:2 FTS). This study demonstrates lower losses of both long- and short-chain PFAS to glass and PET. It also highlights the need for caution when deciding on sample preparatory steps and storage during the analysis of PFAS.

Improved Preservation of Rat Small Intestine Transplantation Graft by Introduction of Mesenchymal Stem Cell-Secreted Fractions.

Segmental grafts from living donors have advantages over grafts from deceased donors when used for small intestine transplantation. However, storage time for small intestine grafts can be extremely short and optimal graft preservation conditions for short-term storage remain undetermined. Secreted factors from mesenchymal stem cells (MSCs) that allow direct activation of preserved small intestine grafts. Freshly excised Luc-Tg LEW rat tissues were incubated in preservation solutions containing MSC-conditioned medium (MSC-CM). Preserved Luc-Tg rat-derived grafts were then transplanted to wild-type recipients, after which survival, injury score, and tight junction protein expression were examined. Luminance for each graft was determined using in vivo imaging. The findings indicated that 30-100 and 3-10kDa fractions of MSC-CM have superior activating effects for small intestine preservation. Expression of the tight-junction proteins claudin-3, and zonula occludens-1 preserved for 24h in University of Wisconsin (UW) solution containing MSC-CM with 50-100kDa, as shown by immunostaining, also indicated effectiveness. Reflecting the improved graft preservation, MSC-CM preloading of grafts increased survival rate from 0% to 87%. This is the first report of successful transplantation of small intestine grafts preserved for more than 24h using a rodent model to evaluate graft preservation conditions that mimic clinical conditions.

Probucol-Ursodeoxycholic Acid Otic Formulations: Stability and In Vitro Assessments for Hearing Loss Treatment

Targeted drug delivery is an ongoing aspect of scientific research that is expanding through the design of micro- and nanoparticles. In this paper, we focus on spray dried microparticles as carriers for a repurposed lipophilic antioxidant (probucol). We characterise the microparticles and quantify probucol prior to assessing cytotoxicity on both control and cisplatin treated hair cells (known as House Ear Institute-Organ of Corti 1; HEI-OC1). The addition of water-soluble polymers to 2% β-cyclodextrin resulted in a stable probucol formulation. Ursodeoxycholic acid (UDCA) used as formulation excipient increases probucol miscibility and microparticle drug content. Formulation characterisations reveals spray drying results in spherical UDCA-drug microparticles with a mean size distribution of ∼5–12 μm. Probucol microparticles show stable short-term storage conditions accounting for only ∼10% loss over seven days. By mimicking cell culture conditions, both UDCA-probucol (67%) and probucol only (82%) microparticles show drug release in the initial two hours. Furthermore, probucol formulations with or without UDCA preserve cell viability and reduce cisplatin-induced oxidative stress. Mitochondrial bioenergetics results in lower basal respiration and non-mitochondrial respiration, with higher maximal respiration, spare capacity, ATP production and proton leak within cisplatin challenged UDCA-probucol groups. Overall, we present a facile method for incorporating lipophilic antioxidant carriers in polymer-based particles that are tolerated by HEI-OC1 cells and show stable drug release, sufficient in reducing cisplatin-induced reactive oxygen species accumulation.

Evaluation of short-term hair follicle storage conditions for maintenance of RNA integrity.

Hair follicles provide an easily accessible tissue for interrogating gene expression for multiple purposes in mammals. RNAlater® is a liquid storage solution that stabilises and preserves cellular RNA, eliminating the need to immediately process or freeze tissue specimens. The manufacturer advises storage of samples at 2-8°C overnight before transfer to -20°C. This study aimed to evaluate RNA integrity in hair follicle samples collected from horses, stabilized in RNAlater®, and stored under three short-term storage conditions. Mane hair samples complete with follicles were collected from four horses at a single time point. Approximately 15 hairs were placed in each of three 2 mL tubes containing 0.75ml RNAlater® solution. Test group A was stored at 4°C for 24-h, then decanted and stored at -20°C. Test groups B and C were stored at 4°C and 19°C (room temperature) respectively for 7 days, then decanted and stored at -20°C. RNA was isolated from all samples and RNA quantity and quality were measured. One-way ANOVA revealed no difference in RNA concentration (A:516 +/-125 ng/ml, B:273+/-93 ng/ml, C:476+/-176 ng/ml;P = 0.2) or quality (A:9.5 +/-0.19, B:9.8+/-0.09, C:9.2+/-0.35 RIN; P = 0.46) between the test groups. There were no group differences in mean Cycle Threshold values from qPCR validation assays confirming high-quality template cDNA. The results suggest that storage of hair follicles for one week in RNAlater® at cool or room temperature conditions will not compromise RNA integrity and will permit extended transport times from remote sampling locations without the need for freezing.

Evaluation of long- and short-term storage conditions and efficiency of a novel microencapsulated Salmonella phage cocktail for controlling Salmonella in chicken meat.

This study aims to assess the stability and activity of using a lyophilization, formulation design and to evaluate their efficiency for controlling Salmonella in chicken meat. The phage-loaded 0.3M sucrose gelatin mixture at 4 and 25°C displayed significantly less phage titer loss (p < 0.05) than the other excipients and liquid phage cocktail in 12months. The results showed that there were significant reductions of Salmonella at the end of the storage in chicken meat for newly prepared phage powder (1.86 log CFU/cm2 and 2.18 log CFU/cm2), lyophilized phage powders stored at 4°C (1.08 log CFU/cm2 and 1.26 log CFU/cm2) and stored at 25°C (0.66 log CFU/cm2 and 1.00 log CFU/cm2) for 10months at MOI 100 and 1000, respectively. The results demonstrated that lyophilized phages in a simple food grade formulation can be successfully stored and might be used in biocontrol of Salmonella in meat.

Quantifying bias introduced by sample collection in relative and absolute microbiome measurements.

To gain insight into the accuracy of microbial measurements, it is important to evaluate sources of bias related to sample condition, preservative method and bioinformatic analyses. There is increasing evidence that measurement of the total count and concentration of microbes in the gut, or 'absolute abundance', provides a richer source of information than relative abundance and can correct some conclusions drawn from relative abundance data. However, little is known about how preservative choice can affect these measurements. In this study, we investigated how two common preservatives and short-term storage conditions impact relative and absolute microbial measurements. OMNIgene GUT OMR-200 yields lower metagenomic taxonomic variation between different storage temperatures, whereas Zymo DNA/RNA Shield yields lower metatranscriptomic taxonomic variation. Absolute abundance quantification reveals two different causes of variable Bacteroidetes:Firmicutes ratios across preservatives. Based on these results, we recommend OMNIgene GUT OMR-200 preservative for field studies and Zymo DNA/RNA Shield for metatranscriptomics studies, and we strongly encourage absolute quantification for microbial measurements.

Dipeptidyl peptidase 4 (DPP4) in fecal samples: validation of the extraction methodology and stability in short-term storage conditions.

This study assesses the clinical relevance of dipeptidyl peptidase 4 (DPP4) membrane exopeptidase as a biomarker of inflammatory bowel disease (IBD). A spike-and-recovery approach of DPP4 in fecal samples was used to compare two different methods for protein extraction, followed by a stability assessment. Fecal samples of healthy volunteers spiked with known concentrations of recombinant DPP4 were processed using a standard manual extraction protocol and the CALEX® protocol. The two methods were compared by quantification of fecal DPP4 by ELISA, followed by Bland-Altman analysis. For the stability assays DPP4 was extracted from fecal samples and stored under different conditions of temperature and time after collection. In general, the levels of spiked DPP4 in stool samples were lower with the manual protocol than in those obtained with the CALEX® method; this trend was corroborated by Bland-Altman analysis. Nonetheless, variability was within the acceptable limits for both protocols. In the stability assessment, no statistically significant differences were found between the results obtained under the different storage conditions. Both manual and CALEX® protocols provided equal extraction ability of DPP4 from stool samples. In addition, DPP4 provided flexibility in terms of sample storage enabling the accurate assessment of samples delivered up to a week before analysis.

Studies on Pollen Morphology, Pollen Vitality and Preservation Methods of Gleditsia sinensis Lam. (Fabaceae)

Gleditsia sinensis Lam. (Fabaceae) is an endemic species in China, which has a wide range of ecological functions and high economic value. G. sinensis belongs to androdioecy, and the stamens of perfect flowers are aborted, meaning that a perfect flower is a functional female flower. Understanding the dynamic process of flowering and the characteristics of pollen morphology effectively determine the viability of pollen vitality, and the suitable conditions for short-term storage of pollen can provide theoretical basis and technical reference for hybrid breeding and germplasm conservation of G. sinensis. In this study, the male plants of G. sinensis in Guiyang area were used as research materials. The flowering dynamic process of male flowers was recorded through field observation. The morphology of pollen was observed and analyzed with a scanning electron microscope (SEM). The germination characteristics of pollen were studied with an in vitro germination method, and the pollen vitality was also determined using four staining methods. The effects of different storage temperatures and water contents on pollen germination rate were discussed. The results showed that the male flowers of G. sinensis had a short, single flowering period, lasting 2–3 days from the opening to the shedding. The dynamic opening process of a single flower was artificially divided into five stages. Pollen grains of G. sinensis are oblate spheroidal, tricolporate with equatorial elongated endoapertures and the sporoderm surface is reticulate. The MTT (Thiazolyl Blue Tetrazolium Bromide) staining method could accurately and quickly determine the pollen vitality of G. sinensis. The highest pollen germination rate was 65.89% ± 3.41%, and the length of the pollen tube was 3.96 mm after cultured in 15% sucrose + 100 mg/L boric acid + 20 mg/L calcium chloride for 24 h. It was necessary to collect the pollen at the big bud stage, which was conducive to improving the efficiency of pollen collection because the pollen had been mature with high pollen vitality at this stage. When it came to pollen preservation, the pollen germination rate was significantly affected by storage time, storage temperature and pollen water content. The pollen still had high vitality after being stored at −80 °C for 30 days when the moisture content of the pollen decreased to 9%, and the pollen germination rate only decreased by 28.84% compared with that before storage. In conclusion, this study has comprehensively and systematically studied the morphology, vitality determination and preservation methods of the pollen of G. sinensis, providing a theoretical basis for the cross regional breeding and the conservation and utilization of germplasm resources.

Understanding the reactivity of magnesium powder subjected to various aging conditions

Metal fuels are emerging as one of the promising alternatives to fossil fuels by providing considerable heat energy without CO2 generation. In particular, magnesium (Mg) is utilized in rocket propellants, fuel cells, and energy storage systems by virtue of its high reactivity, great energy density, and low cost. While its performance degradation when exposed to both short-term and prolonged storage conditions is expected, the clear aging characteristics have not been explained in the past. Two products of hygrothermal aging process of Mg are identified as Mg(OH)2 and MgO, where Mg(OH)2 decomposes at a relatively low temperature for accelerating the oxidation process while MgO tends to desensitize the reactivity of Mg by the surface oxidation of particles. Here, a decreased on-set reaction temperature or enhanced reactivity is observed on relatively short-term aged samples, which is attributed to the formation of Mg(OH)2. For accelerated aging conditions beyond 25 weeks, the oxide film covers the entire particle surface, and thus only the core oxidation could occur, which results in an increase of activation energy. This observation is contrary to the decreased reactivity of metal fuels subjected to aging in general. Moreover, the reactivity based on the stoichiometry is examined by varying the amount of oxygen supplied. The activation energy for long-term aging periods tended to increase under oxygen-rich condition, which can be ascribed to the reaction between Mg(OH)2 and O2. Also, the effects of the generation or the decomposition of various intermediates of the oxidation process can be included. Thus, the present study identified the peculiar enhancement of reactivity of Mg subjected to hygrothermal aging and further clarified the roles of surface and core oxidations as they both affect the reactivity under the oxygen enrichment conditions.

The Effect of Harvest Date and Storage Conditions on the Quality of Remontant Strawberry Cultivars Grown in a Gutter System under Covers

The storage characteristics of three remontant strawberry cultivars San Andreas®, Albion® and Murano® were studied in 2018–2020. The short-term storage conditions (7 days) of strawberries used in the study significantly affected fruit quality indicators. The highest quality of stored strawberries was provided by controlled atmosphere (CA) conditions, as evidenced by the typically highest fruit firmness and the lowest natural loss of strawberry weight. The modified atmosphere packaging (MAP) conditions obtained in the Xtend® bags provided a similar good storage effect for strawberries. Strawberries stored in MAP bags had the lowest respiratory intensity, and their firmness and the percentage of rotten fruit were the same as those stored in a controlled atmosphere. In addition, MAP bags were effective in reducing the weight loss of stored strawberries. The quality of strawberries stored in a conventional air storage (AA) was slightly lower compared to the fruit stored in a controlled and modified atmosphere. This was due to their lower firmness and significantly higher respiration intensity and higher weight loss. In addition, the percentage of rotten fruit, although low, has always been the highest among AA strawberries. The quality of the strawberries also depended on the date of fruit harvest. Strawberries harvested later, compared to those collected earlier, were generally characterized by higher firmness, lower acidity and lower respiration intensity.

Bal Arısı Spermasının Kısa ve Uzun Süreli Saklanmasıyla İlgili Güncel Yaklaşımlar

There are many studies on the storage of honey bee semen. Due to its unique structure, bee semen varies according to the storage period but is suitable for short-term storage conditions. However, in terms of long-term storage conditions, developments in mammals have not been caught. It is known that more studies are needed on this subject. The great advantages of semen storage are its effective role in eliminating the problems of inbreeding after a while in pure breed enterprises, increasing the yield characteristics and preventing gene pollution. To solve these problems, beekeepers have to buy breeding queens from abroad with very high costs, and the expected results cannot always be obtained from the bees brought. Especially considering the genetic risks faced by regional breeds in Turkey, it is necessary to preserve semen, to protect colonies in pure breed enterprises, as well as to develop genetically yield characteristics and to prevent inbreeding. It can also prevent the spread of diseases compared to natural mating by contributing to artificial insemination. By improving the storage conditions of honey bee semen, long-term use of semen from well-known colonies will be possible. Producers will be able to benefit from the advantages of artificial insemination at a cheaper cost, with the semen being sent to distant regions as in mammals under appropriate storage conditions. The purpose of this review is to give information about the storage conditions of honeybee semen.

Influence of short term storage conditions, concentration methods and excipients on extracellular vesicle recovery and function

Extracellular vesicles (EVs) are phospholipid bilayer enclosed vesicles which play an important role in intercellular communication. To date, many studies have focused on therapeutic application of EVs. However, to progress EV applications faster towards the clinic, more information about the physical stability and scalable production of EVs is needed. The goal of this study was to evaluate EV recovery and function after varying several conditions in the isolation process or during storage.Physical stability and recovery rates of EVs were evaluated by measuring EV size, particle and protein yields using nanoparticle tracking analysis, microBCA protein quantification assay and transmission electron microscopy. Western blot analyses of specific EV markers were performed to determine EV yields and purity. EV functionality was tested in an endothelial cell wound healing assay.Higher EV recovery rates were found when using HEPES buffered saline (HBS) as buffer compared to phosphate buffered saline (PBS) during EV isolation. When concentrating EVs, 15 ml spinfilters with a 10 kDa membrane cutoff gave the highest EV recovery. Next, EV storage in polypropylene tubes was shown to be superior compared to glass tubes. The use of protective excipients during EV storage, i.e. bovine serum albumin (BSA) and Tween 20, improved EV preservation without influencing their functionality. Finally, it was shown that both 4 °C and −80 °C are suitable for short term storage of EVs. Together, our results indicate that optimizing buffer compositions, concentrating steps, protective excipients and storage properties may collectively increase EV recovery rates significantly while preserving their functional properties, which accelerates translation of EV-based therapeutics towards clinical application.

A novel canine nuclear magnetic resonance spectroscopy-based metabolomics platform: Validation and sample handling.

Metabolomics has been proven to be an invaluable research tool by providing comprehensive insight into systemic metabolism. However, the lack of scalable and quantitative methods with known reference intervals (RIs) and documented reproducibility has prevented the use of metabolomics in the clinical setting. The objective of this study was to validate the developed quantitative nuclear magnetic resonance (NMR) spectroscopy-based metabolomics platform for canine serum and plasma samples and determine optimal sample handling conditions for its use. Altogether, 8247 canine samples were analyzed using a Bruker's 500MHz NMR spectrometer. Using statistical approaches derived from international guidelines, we studied method precision, measurand stability in various long- and short-term storage conditions, as well as the effect of prolonged contact with red blood cells (RBCs), and differences among blood collection tubes. We also screened interferences with lipemia, hemolysis, and bilirubinemia. The results were compared against routine clinical chemistry methods, and RIs were defined for all measurands. We determined RIs for 123 measurands, most of which were previously unpublished. The reproducibility of the results of the NMR platform appeared generally outstanding, and the integrity of the results can be ensured by following standard blood drawing and processing guidelines. Owing to the advantages of quantitative results, high reproducibility, and scalability, this canine metabolomics platform holds great potential for numerous clinical and research applications to improve canine health and well-being.

Understanding the different emulsification mechanisms of pectin: Comparison between watermelon rind and two commercial pectin sources

In this work, an advanced approach combining small angle X-ray scattering (SAXS) experiments, rheology and confocal laser scanning microscopy was used to explain the different emulsification mechanisms of three pectin sources (pectin extracted from watermelon rind -WRP- and commercial citrus -CP- and apple pectin -AP). Very interestingly, three different emulsification mechanisms were identified, related to the structure and composition of the pectin extracts. WRP had significantly greater emulsifying capacity than commercial CP and AP. This enhanced emulsification ability was mainly ascribed to a combination of its relatively high protein content (mainly acting as the surface-active material), combined with the presence of longer sugar side chains in pectin, further contributing to stabilizing the oil droplets in the emulsions. All these structural features resulted in a reduction in the mean droplet size as the concentration increased, thus, hindering flocculation and coalescence during the short-term storage conditions at 4 °C. In contrast, AP had the lowest emulsification capacity, which was only related to its viscosifying effect (provided by its greater Mw), while CP, having the greatest homogalacturonan content, greatest linearity and a more balanced hydrophilic/hydrophobic character (reflected in the degree of esterification), was able to form a better adsorbed layer at the o/w interphase, although it could not avoid flocculation and creaming at low pectin concentration during refrigerated storage.

Changes in abscisic acid, antioxidant concentration and the activities of antioxidant enzymes in Japanese apricot (Prunus mume) under short-term anoxic storage conditions

Changes in abscisic acid, antioxidant concentration and the activities of antioxidant enzymes in Japanese apricot (<i>Prunus mume</i>) under short-term anoxic storage conditions

Survival and transcriptomic response of Salmonella enterica on fresh-cut fruits

Salmonella enterica is frequently implicated in foodborne disease outbreaks associated with fresh-cut fruits. In the U.S., more than one third of fruit-related outbreaks have been linked to two S. enterica serotypes Newport and Typhimurium. Approximately 80% of fruit-related human salmonellosis cases were associated with tomatoes, cantaloupes and cucumbers. In this study, we investigated the population dynamics of S. Newport and S. Typhimurium on fresh-cut tomato, cantaloupe, cucumber and apple under short-term storage conditions. We further compared the transcriptomic profiles of a S. Newport strain on fresh-cut tomato and cantaloupe using high-throughput RNA-seq. We demonstrated that both S. enterica Newport and Typhimurium survived well on various fresh-cut fruit items under refrigeration storage conditions, independent of inoculation levels. However, S. enterica displayed variable survival behaviors on different types of fruits. For example, at 7 d storage, the population of S. enterica reduced less than 0.2 log (p > 0.05) on fresh-cut tomato and cantaloupe, in contrast to ~0.5 log (p < 0.05) on cucumber and apple. RNA-seq analysis suggested that S. enterica mediates its survival on fresh-cut fruits through differentially regulating genes involved in specific carbon utilization and metabolic pathways. Several known bacterial virulence factors (e.g., pag gene) were found to be differentially regulated on fresh-cut tomato and cantaloupe, suggesting a link between the events of food contamination and subsequent human infection. Findings from this study contribute to a better understanding of S. enterica survival mechanisms on fresh-cut produce.

Effect of fasting status and other pre-analytical variables on quantitation of long-chain fatty acids in red blood cells

Long-chain fatty acids (LCFAs), including omega-3 and omega-6 fatty acids, play essential roles in health maintenance and outcomes. Insufficient intake or the inability to absorb LCFAs from the diet can cause a number of health problems. Evaluation of fatty acid profiles in plasma, serum or red blood cells (RBCs) is routinely used to monitor patients at risk of developing deficiency. Quantitation of LCFAs in RBCs offers advantages over serum/plasma due to low intra-individual variability. Fatty acid composition in RBCs also reflects long-term dietary intake, providing additional information about the patient's nutritional status. However, the literature does not currently address the impact of pre-analytical factors (conditions of RBC collection, sample handling and short-term storage) on LCFA measurements. This study evaluated the effect of several anticoagulants, interferents, different storage conditions and fasting status on quantitation of the twenty-one most abundant LCFAs in RBCs by gas chromatography negative chemical ionization-mass spectrometry (GCNCI-MS). LCFA results were assessed quantitatively (nmol/mL) or as a percent of total. Most common tube types (containing citrate, sodium heparin or EDTA) were all appropriate for blood collection. Whole blood and lysed RBCs were stable at least 24 h at room temperature and up to 7 days refrigerated. Lysed RBCs were also stable for up to three freeze/thaw cycles. The presence of icterus or lipemia did not affect results. LCFAs concentrations in RBCs did not change ~4 h after high-fat intake when the lipid concentration in circulation reaches a peak, while plasma levels of most fatty acids increased up to 40% in response. In summary, RBCs are a reliable sample type for LCFA quantitation in the clinical laboratory. In contrast to plasma or serum, RBCs isolated from non-fasting, hemolyzed or lipemic whole blood specimens are all acceptable for testing. Therefore, RBCs might be a preferable sample type for evaluation of nutritional status of young pediatric patients and in patients with conditions associated with hemolytic anemia or hyperlipidemia.

Sperm Storage and Artificial Insemination in Honey Bees

To obtain the expected yield from a honeybee colony, queen bee is required to have high egg capacity, the colony is also expected to be resistant to diseases, to have a low tendency for swarming, to be resistant to looting, and to be able to adapt to the climatic conditions of the region. In this context, it is important to protect the pure bee races and to improve them by conducting breeding research. To maintain the high yield aspect of honeybee colony, the queen bee needs to be replaced at most every 2 years. Queen bee becomes adult in as short as 16 days after hatching, and mating is realized in the air outside the colony, which makes it difficult to preserve the genetic line of the queen bee. At this point, artificial insemination and protection of gene resources become important. Honey bee (Apis mellifera L.) sperm can be stored for short and long periods. However, due to the delicate structure and biology of honey bee sperm, the high success rate in mammals could not be achieved in storing bee sperm. Due to the genetic damage exposed during the freezing of honey bee sperm, long-term storage difficulties are experienced. In addition, the concentration, motility and viability of spermatozoa decrease due to the short-term storage and storage conditions. In the breeding studies in the same region, after a period of time, gene resources decrease, and accordingly, the risk of inbreeding occurs. Instead of replacing the bee colonies that are at risk of inbreeding, a new different genome addition to the colony whose current yield characteristics are known can be made through the sperm storage of other colonies. Thanks to the long-term storage of sperm, long-period genetic studies can be carried out as in mammals, which is important for improving yield characteristics genetically. In addition, the long-term storage of honeybee sperm is a hope for the protection of regional races that are in danger of extinction due to unconsciousness and improper breeding policies.

Evaluation of short-term storage prior to analysis of vitamin D3 and metabolites in human serum by liquid chromatography coupled to tandem mass spectrometry

Vitamin D has been widely determined in clinical trials to elucidate its biochemical involvement in a great number of pathologies. The analysis of vitamin D and its hydroxymetabolites in biofluids such as serum or plasma is a challenging task due to limitations associated to the low concentrations of some metabolites (typically, dihydroxymetabolites), methodological interferences, and the low stability of the compounds. Among these limitations, efforts have been targeted at optimizing instrumental improvements to develop more sensitive and selective methods, while the stability of vitamin D and metabolites has not been exhaustively evaluated. In this research, several aspects regarding the short-term storage conditions of human serum have been studied to evaluate their influence on the determination of vitamin D3 and metabolites. An experimental plan has been applied to assess the influence of two relevant parameters: the storage temperature for a period of two months and the number of freeze–thaw cycles. The storage temperature affected in a different manner to vitamin D3 and its metabolites, being vitamin D3 and 1,25-dihydroxyvitamin D3 the two analytes more affected by this parameter. Concerning the freeze–thaw cycles, this variable must be limited to two cycles owing to its significant influence on the variability for quantitation of dihydroxymetabolites in human serum. Finally, lyophilization was also tested to check if serum concentrations of vitamin D3 and its metabolites were affected by this preprocessing step. The results revealed that only vitamin D3 experienced a decrease in serum concentration after two months, which does not constitute a real problem as vitamin D3 is not currently a crucial parameter to be determined in clinical trials due to its scant biological activity.