Evaluation of Simultaneous Growth of Escherichia coli O157:H7, Salmonella spp., and Listeria monocytogenes in Ground Beef Samples in Different Growth Media.

Several multiplex approaches for the simultaneous detection of pathogens in food have been developed in recent years, but the use of a single enrichment medium remains a problem. In this study, six enrichment broths (five non-selective media, tryptic soy broth (TSB), brain heart infusion broth (BHI), buffered peptone water (BPW), universal pre-enrichment broth (UPB), no. 17 broth, and a selective, Salmonella Escherichia Listeria broth (SEL)), were studied for the simultaneous detection of E. coli O157:H7, Salmonella spp., and L. monocytogenes, to validate the suitable enrichment broth to be used for the detection methods. Different ratios of E. coli O157:H7, Salmonella spp., and L. monocytogenes were used. Almost all non-selective broths evaluated in this study showed similar growth parameters and profiles among each other. The only selective enrichment broth under analysis (SEL) showed distinct growth features compared to the non-selective media, allowing for a slower but balanced growth of the three pathogens, which could be beneficial in preventing the overgrowth of fast-growing bacteria. In addition, when tested in ground beef samples, SEL broth seems to be the most distinctive medium with a balanced growth pattern observed for the three pathogens. Overall, this study is intended to provide the basis for the selection of suitable enrichment broths according to the technology detection to be used, the desired time of enrichment, and the expected balanced concentration of pathogens.

Addressing bioreactor hiPSC aggregate stability, maintenance and scaleup challenges using a design of experiment approach.

BACKGROUND: Stem cell-derived therapies hold the potential for treatment of regenerative clinical indications. Static culture has a limited ability to scale up thus restricting its use. Suspension culturing can be used to produce target cells in large quantities, but also presents challenges related to stress and aggregation stability. METHODS: Utilizing a design of experiments (DoE) approach in vertical wheel bioreactors, we evaluated media additives that have versatile properties. The additives evaluated are Heparin sodium salt (HS), polyethylene glycol (PEG), poly (vinyl alcohol) (PVA), Pluronic F68 and dextran sulfate (DS). Multiple response variables were chosen to assess cell growth, pluripotency maintenance and aggregate stability in response to the additive inputs, and mathematical models were generated and tuned for maximal predictive power. RESULTS: Expansion of iPSCs using 100 ml vertical wheel bioreactor assay for 4 days on 19 different media combinations resulted in models that can optimize pluripotency, stability, and expansion. The expansion optimization resulted in the combination of PA, PVA and PEG with E8. This mixture resulted in an expansion doubling time that was 40% shorter than that of E8 alone. Pluripotency optimizer highlighted the importance of adding 1% PEG to the E8 medium. Aggregate stability optimization that minimizes aggregate fusion in 3D culture indicated that the interaction of both Heparin and PEG can limit aggregation as well as increase the maintenance capacity and expansion of hiPSCs, suggesting that controlling fusion is a critical parameter for expansion and maintenance. Validation of optimized solution on two cell lines in bioreactors with decreased speed of 40 RPM, showed consistency and prolonged control over aggregates that have high frequency of pluripotency markers of OCT4 and SOX2 (> 90%). A doubling time of around 1-1.4 days was maintained after passaging as clumps in the optimized medium. Controlling aggregate fusion allowed for a decrease in bioreactor speed and therefore shear stress exerted on the cells in a large-scale expansion. CONCLUSION: This study resulted in a control of aggregate size within suspension cultures, while informing about concomitant state control of the iPSC state. Wider application of this approach can address media optimization complexity and bioreactor scale-up challenges.

Quantification of amino acids secreted by yeast cells by hydrophilic interaction liquid chromatography-tandem mass spectrometry.

Monitoring the levels of amino acids (AAs) in biological cell cultures provides key information to understand the regulation of cell growth and metabolism. Saccharomyces cerevisiae can naturally excrete AAs, making accurate detection and determination of amino acid levels within the cultivation medium pivotal for gaining insights into this still poorly known process. Given that most AAs lack ultraviolet (UV) chromophores or fluorophores necessary for UV and fluorescence detection, derivatization is commonly utilized to enhance amino acid detectability via UV absorption. Unfortunately, this can lead to drawbacks such as derivative instability, labor intensiveness, and poor reproducibility. Hence, this study aimed to develop an accurate and stable hydrophilic interaction liquid chromatography-tandem mass spectrometry analytical method for the separation of all 20 AAs within a short 17-min run time. The method provides satisfactory linearity and sensitivity for all analytes. The method has been validated for intra- and inter-day precision, accuracy, recovery, matrix effect, and stability. It has been successfully applied to quantify 20 AAs in samples of yeast cultivation medium. This endeavor seeks to enhance our comprehension of amino acid profiles in the context of cell growth and metabolism within yeast cultivation media.

In search of the best method to detect carriage of carbapenem-resistant Pseudomonas aeruginosa in humans: a systematic review.

BACKGROUND: Detection of carbapenem-resistant Pseudomonas aeruginosa (CR-PA) in humans is important to prevent transmission. However, the most optimal culture method to detect CR-PA is unknown. This systematic review aims to determine which culture method is most sensitive and which culture methods are used to detect CR-PA in humans. Second, to establish the most feasible culture method taking into account the turnaround time (TAT), and third, to provide an overview of the sampling sites used to detect carriage. METHODS: We systematically searched the electronic databases Embase, Medline Ovid, Cochrane, Scopus, CINAHL, and Web of Science until January 27, 2023. All diagnostic accuracy studies comparing two or more culture methods to detect CR-PA and recent outbreak or surveillance reports on CR-PA carriage or infection in humans, which describe culture methods and their results, were eligible for inclusion. We used QUADAS-2 guideline for diagnostic accuracy studies and the STROBE or ORION guideline for outbreak-surveillance studies to assess the risk of bias. RESULTS: Six diagnostic accuracy studies were included. An enrichment broth was found to increase the detection of CR-PA. Using an enrichment broth extended the TAT by 18-24 h, yet selective media could reduce the TAT by 24 h compared to routine media. In total, 124 outbreak-surveillance studies were included, of which 17 studies with surveillance samples and 116 studies with clinical samples. In outbreak-surveillance studies with surveillance samples, perianal, rectal swabs or stools were the most common sampling site/specimen (13/17, 76%). A large variety was observed in whether and which kind of enrichment broth and selective media were used. CONCLUSIONS: We found a benefit of using an enrichment step prior to inoculation of the material onto selective media for the detection of CR-PA. More research is needed to determine the most sensitive sampling site and culture method. TRAIL REGISTRATION: This study was registered in the PROSPERO International prospective register of systematic reviews (registration number: CRD42020207390, http://www.crd.york.ac.uk/PROSPERO/display_record.asp?ID=CRD42020207390 ).

Super-Transparent Soil for In Situ Observation of Root Phenotypes.

Transparent soil (TS) presents immense potential for root phenotyping due to its ability to facilitate high-resolution imaging. However, challenges related to transparency, mechanical properties, and cost hinder its development. Herein, we introduce super-transparent soil (s-TS) prepared via the droplet method using low acyl gellan gum and hydroxyethyl cellulose crosslinked with magnesium ions. The refractive index of the hydroxyethyl cellulose solution (1.345) closely aligns with that of water (1.333) and the low acyl gellan gum solution (1.340), thereby significantly enhancing the transmittance of hydrogel-based transparent soil. Optimal transmittance (98.45%) is achieved with polymer concentrations ranging from 0.8 to 1.6 wt.% and ion concentrations between 0.01 and 0.09 mol·L-1. After 60 days of plant cultivation, s-TS maintains a transmittance exceeding 89.5%, enabling the detailed visualization of root growth dynamics. Furthermore, s-TS exhibits remarkable mechanical properties, withstanding a maximum compressive stress of 477 kPa and supporting a maximum load-bearing depth of 186 cm. This innovative approach holds promising implications for advanced root phenotyping studies, fostering the investigation of root heterogeneity and the development of selective expression under controlled conditions.

Analysis of current technology status for the industrialization of cultured meat.

Cultured meat is expected to become an important material for future food production; however, contrary to initial expectations, the full-scale industrialization of cultured meat is slow and the actual level and opened technology amount is very limited. This study reviews the publicly available technologies of cultured meat and suggests future developmental directions and research agenda. As a result of analyzing papers, patents, and press releases published over the past 10 years, it was found that cultured meat production technology is still at the prototype production level. This is because most papers published are about culture medium and scaffold development, culture conditions, and there is almost no research on finished cultured meat products. Worldwide, most of the filed patents are for producing cultured meat principles; most of them do not use food-grade materials and are not economically feasible for industrialization. Therefore, future research on the industrialization of cultured meat should focus on effective acquisition technologies for satellite cells; cell lineage and undifferentiated state maintenance technologies; the development of serum-free media and culture devices; the prevention of genetic modification, safety verification, and mass production. Furthermore, basic research on mechanisms and influencing factors related to cultured meat production is warranted.

Optimizing Cucumis sativus seedling vigor: the role of pistachio wood vinegar and date palm compost in nutrient mobilization.

BACKGROUND: The goal of this research is to enhance the quality of cucumber seedlings grown in greenhouses by experimenting with various soilless culture mediums (CMs) and the application of pistachio wood vinegar (WV). The experimental setup was designed as a factorial experiment within a randomized complete block design (RCBD), in greenhouse conditions featuring three replications to assess the effects of different culture media (CMs) and concentrations of pistachio wood vinegar (WV) on cucumber seedling growth. Cucumber seeds were planted in three CMs: coco peat-peat moss, coco peat-vermicompost, and date palm compost-vermicompost mixed in a 75:25 volume-to-volume ratio. These were then treated with pistachio WV at concentrations of 0, 0.5, and 1%, applied four times during irrigation following the emergence of the third leaf. RESULTS: The study revealed that treating seedlings with 0.5% WV in the date palm compost-vermicompost CM significantly enhanced various growth parameters. Specifically, it resulted in a 90% increase in shoot fresh mass, a 59% increase in shoot dry mass, an 11% increase in root fresh mass, a 36% increase in root dry mass, a 65% increase in shoot length, a 62% increase in leaf area, a 25% increase in stem diameter, a 41% increase in relative water content (RWC), and a 6% improvement in membrane stability index (MSI), all in comparison to untreated seedlings grown in coco peat-peat moss CM. Furthermore, chlorophyll a, b, total chlorophyll, and carotenoid levels were 2.3, 2.7, 2.6, and 2.7 times higher, respectively, in seedlings treated with 0.5% WV and grown in the date palm compost-vermicompost CM, compared to those treated with the same concentration of WV but grown in coco peat-peat moss CM. Additionally, the Fv/Fm ratio saw a 52% increase. When plant nutrition was enhanced with the date palm compost-vermicompost CM and 1% WV, auxin content rose by 130% compared to seedlings grown in coco peat-peat moss CM and treated with 0.5% WV. CONCLUSIONS: The study demonstrates that using 0.5% WV in conjunction with date palm compost-vermicompost CM significantly betters the quality of cucumber seedlings, outperforming other treatment combinations.

Expression of miRNA from spent pre-implantation embryos culture media.

This study examines the expression of three microRNAs (hsa-miR-661, hsa-miR-21-5p, hsa-miR-372-5p) in spent pre-implantation embryos culture media to identify possible new non-invasive biomarkers of embryo competence, predictive of development to the blastocyst stage. A preliminary analysis on 16 patients undergoing IVF cycles was performed by collecting and stored spent culture media on the fifth/sixth day of embryo culture. Expression of miRNAs was evaluated according to the embryos' fate: 1) NE/DG: non-evolved or degenerate embryos; 2) BLOK: embryos developed to the blastocyst stage. Preliminary results revealed a higher miRNAs expression in NE/DG spent media. To elucidate the roles of these miRNAs, we employed a robust bioinformatics pipeline involving: 1) in-silico miRNA Target Prediction using RNAHybrid, which identified the most-likely gene targets; 2) Construction of a Protein-Protein Interaction network via GeneMania, linking genes with significant biological correlations; 3) application of modularity-based clustering with the gLay app in Cytoscape, resulting in three size-adapted subnets for focused analysis; 4) Enrichment Analysis to discern the biological pathways influenced by the miRNAs. Our bioinformatics analysis revealed that hsa-miR-661 was closely associated with pathways regulating cell shape and morphogenesis of the epithelial sheet. These data suggest the potential use of certain miRNAs to identify embryos with a higher likelihood of developing to the blastocyst stage. Further analysis will be necessary to explore the reproducibility of these findings and to understand if miRNAs here investigated can be used as biomarkers for embryo selection before implantation into the uterus or if they may be reliable predictors of IVF outcome.

Use of spectroscopic process analytical technology for rapid quality evaluation during preparation of CHO cell culture media.

Media preparation parameters contribute significantly to media quality, cell culture performance, productivity, and product quality. Establishing proper media preparation procedures is critical for ensuring a robust CHO cell culture process. Process analytical technology (PAT) enables unique ways to quantify assessments and improve media quality. Here, cell culture media were prepared under a wide range of temperatures (40-80°C) and pH (7.6-10.0). Media quality profiles were compared using three real-time PATs: Fourier-transform infrared (FTIR) spectroscopy, Raman spectroscopy, and excitation-emission matrix (EEM) spectroscopy. FTIR and Raman spectroscopies identified shifts in media quality under high preparation temperature (80°C) and at differing preparation pH which negatively impacted monoclonal antibody (mAb) production. In fed-batch processes for production of three different mAbs, viable cell density (VCD) and cell viability were mostly unaffected under all media preparation temperatures, while titer and cell specific productivity of mAb decreased when cultured in basal and feed media prepared at 80°C. High feed preparation pH alone was tolerated but cell growth and productivity profiles deviated from the control condition. Further, charge variants (main, acidic, basic species) and glycosylation (G0F, afucosylation, and high mannose) were examined. Statistically significant differences were observed for one or more of these quality attributes with any shifts in media preparation. In this study, we demonstrated strong associations between media preparation conditions and cell growth, productivity, and product quality. The rapid evaluation of media by PAT implementation enabled more comprehensive understanding of different parameters on media quality and consequential effects on CHO cell culture.

Impact of culture media pre-equilibration methods on embryo development.

This study investigates the influence of four culture media pre-equilibration methods on embryo development and clinical pregnancy outcomes. The methods are as follows: Method A involved covering media with fresh mineral oil in humid-type incubators for 24 h. Method B replicated Method A in dry-type incubators. Method C utilized pre-equilibrated (humidified) mineral oil to cover the media, also in humid-type incubators for 24 h. Method D followed the same process as Method C but in dry-type incubators. Subsequently, media from all groups were transferred to dry-type incubators for 72 h. Osmolality was measured at 24, 48, 72, and 96 h. For G1 PLUS, no significant differences were observed among groups at 24, 48, and 72 h. However, at 96 h, Groups B and D exhibited significantly higher osmolality than Groups A and C (A vs B, p = 0.043; A vs D: p = 0.046; B vs C, p = 0.043; C vs D, p = 0.046). No significant variations were found between Groups A and C or B and D. Similar results were obtained for G2 PLUS. A retrospective analysis of embryo development and clinical outcomes using Methods A revealed significant improvements in good blastocysts and available embryos compared with Method B for all (p = 0.005 and 0.004) and IVF cycles (p = 0.025 and 0.017). Method A also enhanced blastocyst formation in ICSI cycles (p = 0.017). However, clinical pregnancy outcomes did not significantly differ between Methods A and B. Pre-equilibrating culture media overnight in humid-type incubators, even when covered with fresh mineral oil, significantly mitigates osmolality rise and improves embryo development potential during embryo culture in dry-type incubators.

New parameters for in vitro development of cell lines of the species Astyanax bimaculatus (Linnaeus, 1758) and Geophagus proximus (Castelnau, 1855).

Intending to compare in vitro cell growth in different conditions, we established cell cultures using fin biopsies of two freshwater fishes, Astyanax bimaculatus and Geophagus proximus. Three different culture media (Leibovitz-L-15, Dulbecco's Modified Eagle Medium [DMEM], and 199) were employed, with or without the addition of AmnioMax, maintaining a standard temperature of 29°C. Based on the results obtained, we standardized a cell growth protocol in which medium 199 was less efficient for both species. Notably, G. proximus cells exhibited superior proliferation in DMEM and L-15 media, whereas A. bimaculatus cells demonstrated better parameters exclusively in the DMEM medium. Successful subculturing of cells with good proliferation index was observed, accompanied by preserved morphological characteristics. Therefore, the methodology outlined in this study represents an advancement in establishing fish cell cultures.

Evolution of Minimally Invasive and Non-Invasive Preimplantation Genetic Testing: An Overview.

Preimplantation genetic testing (PGT) has become a common supplementary diagnοstic/testing tοol for in vitro fertilization (ΙVF) cycles due to a significant increase in cases of PGT fοr mοnogenic cοnditions (ΡGT-M) and de novο aneuplοidies (ΡGT-A) over the last ten years. This tendency is mostly attributable to the advancement and application of novel cytogenetic and molecular techniques in clinical practice that are capable of providing an efficient evaluation of the embryonic chromosomal complement and leading to better IVF/ICSI results. Although PGT is widely used, it requires invasive biopsy of the blastocyst, which may harm the embryo. Non-invasive approaches, like cell-free DNA (cfDNA) testing, have lower risks but have drawbacks in consistency and sensitivity. This review discusses new developments and opportunities in the field of preimplantation genetic testing, enhancing the overall effectiveness and accessibility of preimplantation testing in the framework of developments in genomic sequencing, bioinformatics, and the integration of artificial intelligence in the interpretation of genetic data.

The addition of mammalian cell culture medium impacts nanoparticle toxicity in zebrafish.

Engineered nanomaterials (ENMs) are ubiquitous in contemporary applications, yet their environmental and human health impacts remain inadequately understood. This study addresses the challenge of identifying potential risks associated with ENM exposure by highlighting the significant variability in existing research methodologies. Without a systematic collection of toxicological data that encompasses standardized materials, relevant platforms, and assays, the task of identifying potential risks linked to ENM exposure becomes an intricate challenge. In vitro assessments often use media rich in ionic species, such as RPMI and fetal bovine serum (FBS). Zebrafish embryos, known to develop normally in low-ionic environments, were exposed to Cerium Oxide, Zinc Oxide, and Graphene Oxides in different media at varying concentrations. Here, we discovered that zebrafish embryos tolerated a mix of 80 % RPMI, 2 % FBS, and 1 % antibiotic cocktail. The results revealed that adverse effects observed in zebrafish with certain nanomaterials in Ultra-Pure (UP) water were mitigated in cell culture medium, emphasizing the importance of revisiting previously considered non-toxic materials in vitro. The zebrafish results underscore the importance of utilizing a multidimensional in vivo platform to gauge the biological activity of nanomaterials accurately.

Metabolomic profiling of embryo culture media in patients with repeated implantation failure during assisted reproductive technology cycles.

Objective: This study investigated the metabolic status of the spent culture media from embryos of patients with repeated implantation failure (RIF) undergoing in vitro fertilization-intracytoplasmic sperm injection cycles in comparison with the embryos from healthy fertile women. Methods: Metabolite levels in spent culture media were assessed and compared between embryos from RIF patients (n=35) and oocyte donors as controls (n=15). Protein levels of insulin-like growth factor 1 (IGF-1) were determined using Western blotting. Concentrations of glucose, pyruvate, and lactate were measured using spectrophotometry. Ionic colorimetric assay kits were utilized to analyze the concentrations of sodium, chloride, calcium, and magnesium ions. High-performance liquid chromatography was employed to measure the concentrations of glutamic acid, aspartic acid, methionine, phenylalanine, and histidine. Results: Glucose consumption and lactate secretion were higher in the control group than in the RIF group. The magnesium concentration was significantly higher in the control group than in the RIF group, but glutamic acid and aspartic acid concentrations were lower in the control group than in the RIF patients (p<0.05). The levels of IGF-1, sodium, calcium, chloride, methionine, histidine, and phenylalanine did not show statistically significant differences between the two groups. Conclusion: The metabolic profile of the culture medium of the embryos in the RIF group differed from that of the control group. These findings suggest potential factors that may affect implantation capacity in RIF patients and provide a new perspective on embryo selection.

Sunken Riches: Ascomycete Diversity in the Western Mediterranean Coast through Direct Plating and Flocculation, and Description of Four New Taxa.

The Mediterranean Sea stands out as a hotspot of biodiversity, whose fungal composition remains underexplored. Marine sediments represent the most diverse substrate; however, the challenge of recovering fungi in culture hinders the precise identification of this diversity. Concentration techniques like skimmed milk flocculation (SMF) could represent a suitable solution. Here, we compare the effectiveness in recovering filamentous ascomycetes of direct plating and SMF in combination with three culture media and two incubation temperatures, and we describe the fungal diversity detected in marine sediments. Sediments were collected at different depths on two beaches (Miracle and Arrabassada) on the Spanish western Mediterranean coast between 2021 and 2022. We recovered 362 strains, and after a morphological selection, 188 were identified primarily with the LSU and ITS barcodes, representing 54 genera and 94 species. Aspergillus, Penicillium, and Scedosporium were the most common genera, with different percentages of abundance between both beaches. Arrabassada Beach was more heterogeneous, with 42 genera representing 60 species (Miracle Beach, 28 genera and 54 species). Although most species were recovered with direct plating (70 species), 20 species were exclusively obtained using SMF as a sample pre-treatment, improving our ability to detect fungi in culture. In addition, we propose three new species in the genera Exophiala, Nigrocephalum, and Queenslandipenidiella, and a fourth representing the novel genus Schizochlamydosporiella. We concluded that SMF is a useful technique that, in combination with direct plating, including different culture media and incubation temperatures, improves the chance of recovering marine fungal communities in culture-dependent studies.

Current technology and industrialization status of cell-cultivated meat.

Interest and investment in cultivated meat are increasing because of the realization that it can effectively supply sufficient food resources and reduce the use of livestock. Nevertheless, accurate information on the specific technologies used for cultivated meat production and the characteristics of cultivated meat is lacking. Authorization for the use of cultivated meat is already underway in the United States, Singapore, and Israel, and other major countries are also expected to approve cultivated meat as food once the details of the intricate process of producing cultivated meat, which encompasses stages such as cell proliferation, differentiation, maturation, and assembly, is thoroughly established. The development and standardization of mass production processes and safety evaluations must precede the industrialization and use of cultivated meat as food. However, the technology for the industrialization of cultivated meat is still in its nascent stage, and the mass production process has not yet been established. The mass production process of cultivated meat may not be easy to disclose because it is related to the interests of several companies or research teams. However, the overall research flow shows that equipment development for mass production and cell acquisition, proliferation, and differentiation, as well as for three-dimensional production supports and bioreactors have not yet been completed. Therefore, additional research on the mass production process and safety of cultivated meat is essential. The consumer's trust in the cultivated meat products and production technologies recently disclosed by some companies should also be analyzed and considered for guiding future developments in this industry. Furthermore, close monitoring by academia and the government will be necessary to identify fraud in the cultivated meat industry.

Sertoli cell-conditioned medium can improve blood-testis-barrier function and spermatogenesis in azoospermia mice induced by scrotal hyperthermia: An experimental study.

Background: An increase in the temperature of the testis is associated with damage to the epithelium of seminiferous tubules and disruption of sperm production. Objective: The current study aimed to investigate the effect of the Sertoli cell-conditioned medium (SCCM) on the blood-testis-barrier associated genes and spermatogenesis process following scrotal hyperthermia. Materials and Methods: In this experimental study, 40 adult NMRI mice (8 wk, 25-30 gr) were allocated into 4 groups: I) control, II) DMEM (10 µl Dulbecco's Modified Eagle Medium), III) scrotal hyperthermia, and IV) scrotal hyperthermia+SCCM (10 µl SCCM). Hyperthermia was induced by placing the mice scrotum in water at 43 C for 20 min every other day for 10 days. Mice were treated every other day for 5 wk. Then the animals were euthanized, and the tails of epididymis were removed to analyze sperm parameters, testis were taken for stereological assessment, reactive oxygen spices and glutathione levels, and the expression of Ocln, Gja1, Cdh2, and Itgb1. Results: The results of sperm analysis indicated that SCCM-treated mice significantly increased sperm count and motility and reduced DNA fragmentation. In addition, histological and molecular findings showed that the volume of testicular tissue, the number of germ cells, the glutathione level, and the expression of Ocln, Gja1, Cdh2, and Itgb1 genes were significantly increased in the SCCM-treated mice. Conclusion: Findings suggest that growth factors of SCCM stimulate the proliferation and differentiation of germ cells through paracrine effects and upregulate the blood-testis-barrier-associated genes in mice subjected to scrotal hyperthermia.

Metabolomic changes in culture media with varying passage numbers of pig muscle stem cell culture for cultured meat production.

Selecting the primary cells in an optimal state for cultured meat production is a crucial challenge in commercializing cultured meat. We investigated the metabolomic changes in culture media according to passage numbers for indirectly assessing the state of primary cells. Pig skeletal muscle stem cells (PSCs) harvested from the biceps femoris muscles of 7-d-old crossbred pigs (Landrace × Yorkshire × Duroc, LYD) were used for cell characterization. Fresh media (FM) and spent media (SM) of PSCs during passages 1 to 3 in vitro culture were prepared for metabolomics analysis. SM was collected on the third day of proliferation for each passage of PSCs. Cell characterization analysis revealed that the proliferation rate was highest at passage 2; however, a significant loss of expression of myogenic marker genes was observed at passage 3. Based on metabolomic profiles of culture media, FM and SM groups (SM1, SM2, and SM3) were clearly separated by partial least squares-discriminant analysis. A total of seven differentially abundant metabolites (DAMs) were identified from FM and SM for each passage, based on the following criteria: P < 0.05, fold change > 1.5 or < 0.66, and a variable importance in projection score > 1.5. All seven DAMs and their interconnected metabolites might be primarily used as substrates for energy production and most of them were relatively abundant in SM3. Among the seven DAMs, the three potential biomarkers (γ-glutamyl-L-leucine, cytosine, and ketoleucine), which showed significant changes exclusively in SM3, each had an area under the curve value of 1. Therefore, monitoring the levels of these key metabolites in culture media could serve as a quality control measure for cultured meat production by enabling the indirect detection of suboptimal PSCs based on their proliferation ability.

Enhancing chlamydospore production in Duddingtonia flagrans on solid substrate: The impact of mannitol and varied cultivation conditions.

Duddingtonia flagrans is a nematophagous fungus which has shown promising results as a non-chemical parasitic control tool. The fungus disrupts the parasite's life cycle by trapping larvae in the environment through the networks generated from chlamydospores, thus preventing the reinfection of animals. One barrier to the development of a commercial product using this tool is the need to increase chlamydospore production in the laboratory for its administration to livestock. The purpose of this study was to evaluate the addition of mannitol to an enriched culture medium and the effect of adverse cultivation conditions on chlamydospore production. D. flagrans was cultivated on Petri dishes with corn agar for 4 weeks at 27 °C and 70% relative humidity (RH). Four groups were then formed, all with Sabouraud agar as a base, to which different growth inducers were added: GSA (glucose Sabouraud agar), GSA-MI (glucose Sabouraud agar + meso inositol), GSA-E (enriched glucose Sabouraud agar), and AE-M (enriched agar + mannitol). After 4 weeks, chlamydospores were recovered by washing the surface of each plate with distilled water and then quantified. The medium that yielded the highest amount of chlamydospores was subjected to different cultivation conditions: NC (normal conditions): 70% RH and 27 °C, AC (adverse conditions) 1: 20% RH and 40 °C, CA2: 60% RH and 27 °C, and CA3: 55% RH and 24 °C. It was determined that mannitol increases chlamydospore production (65x106 chlamydospores/plate), and when reducing humidity by 10% under cultivation conditions it resulted in an approximately 10% increase in chlamydospore production compared to the control group. These results suggest that the addition of polyols, as well as its cultivation under certain environmental conditions, can improve chlamydospore production on a laboratory scale.

Culture media affect sex after IVF treatment-a detailed analysis of explanatory variables.

PURPOSE: The purpose of this study was to provide a detailed analysis of clinical and laboratory factors associated with skewed secondary sex ratio (SSR) after ART. METHOD: Retrospective cohort study of embryos resulting in live births, from frozen and fresh single blastocyst transfers. Embryos were cultured in either G-TL (n = 686) or Sage media (n = 685). Data was analyzed using a multivariate logistic regression model and a mixed model analysis. RESULTS: Significantly more male singletons were born after culture in Sage media compared to G-TL media (odds ratio (OR) 1.34, 95% CI (1.05, 1.70), P = 0.02). Inner cell mass grade B vs A (OR 1.36 95% CI (1.05, 1.76), P = 0.02) and one previous embryo transfer (OR 1.49, 95% CI (1.03, 2.16), P = 0.03) were associated with a significantly higher probability of male child at birth. Factors associated with a reduced probability of male child were expansion grade 3 vs 5 (OR 0.66, 95% CI (10.45, 0.96), P = 0.03) and trophectoderm grade B vs A (OR 0.57, 95% CI (0.44, 0.74), P = 0.00). Male embryos developed significantly faster in Sage media compared to G-TL media for the stages of blastocyst (- 1.12 h, 95% CI (- 2.12, - 0.12)), expanded blastocyst (- 1.35 h, 95% CI (- 2.34, - 0.35)), and hatched blastocyst (- 1.75 h, 95% CI (- 2.99, - 0.52)). CONCLUSION: More male children were born after culture in Sage media compared to G-TL media. Male embryo development was affected by culture media. Our observations suggest that culture media impact male embryo quality selectively, thus potentially favoring the selection of male embryos.