Effects of Melatonin, GM-CSF, IGF-1, and LIF in Culture Media on Embryonic Development: Potential Benefits of Individualization.

The implantation of good-quality embryos to the receptive endometrium is essential for successful live birth through in vitro fertilization (IVF). The higher the quality of embryos, the higher the live birth rate per cycle, and so efforts have been made to obtain as many high-quality embryos as possible after fertilization. In addition to an effective controlled ovarian stimulation process to obtain high-quality embryos, the composition of the embryo culture medium in direct contact with embryos in vitro is also important. During embryonic development, under the control of female sex hormones, the fallopian tubes and endometrium create a microenvironment that supplies the nutrients and substances necessary for embryos at each stage. During this process, the development of the embryo is finely regulated by signaling molecules, such as growth factors and cytokines secreted from the epithelial cells of the fallopian tube and uterine endometrium. The development of embryo culture media has continued since the first successful human birth through IVF in 1978. However, there are still limitations to mimicking a microenvironment similar to the reproductive organs of women suitable for embryo development in vitro. Efforts have been made to overcome the harsh in vitro culture environment and obtain high-quality embryos by adding various supplements, such as antioxidants and growth factors, to the embryo culture medium. Recently, there has been an increase in the number of studies on the effect of supplementation in different clinical situations such as old age, recurrent implantation failure (RIF), and unexplained infertility; in addition, anticipation of the potential benefits from individuation is rising. This article reviews the effects of representative supplements in culture media on embryo development.

In vitro maturation in synthetic oviductal fluid increases gene expression associated with quality and lipid metabolism in bovine oocytes.

Traditionally, in vitro oocyte and embryo culture progresses through a series of varying culture medium. To investigate simplifying the in vitro production of bovine cumulus-oocyte complexes (COCs), this study used synthetic oviductal fluid (SOF) supplemented with conjugated linoleic acid (CLA). Special interest was placed on gene expression linked to lipid metabolism and oocyte maturation. COCs were matured in different media: Medium 199 (M199 group), M199 with 100 µM CLA (M199 + CLA group), SOF (SOF group), and SOF with 100 µM CLA (SOF + CLA group). COCs matured with SOF showed a higher relative abundance of mRNA of quality indicators gremlin 1 (GREM1) and prostaglandin-endoperoxide synthase 2 (PTGS2) in oocytes, and GREM1 in cumulus cells compared with in the M199 group. SOF medium COCs had a higher relative abundance of fatty acid desaturase 2 (FADS2) compared with the M199 group, which is essential for lipid metabolism in oocytes. Furthermore, the abundance of stearoyl-coenzyme A desaturase 1 (SCD1) in oocytes matured with SOF was not influenced by the addition of CLA, whereas the relative abundance of SCD1 was reduced in M199 medium with CLA. We concluded that maturation in SOF medium results in a greater abundance of genes linked to quality and lipidic metabolism in oocytes, regardless of the addition of CLA.

The influence of growth medium on the photodynamic susceptibility of Aggregatibacter actinomycetemcomitans to antimicrobial blue light.

The aim of this study was to investigate whether antimicrobial blue light (aBL) can cause the death of Aggregatibacter actinomycetemcomitans (A.a) and to determine the influence of different culture media, specifically brain heart infusion and blood agar, on bacterial survival fraction. An LED emitting at 403 ± 15 nm, with a radiant power of 1W, irradiance of 588.2 mW/cm2, and an irradiation time of 0 min, 1 min, 5 min, 10 min, 30 min, and 60 min, was used. The plates were incubated in microaerophilic conditions at 37 °C for 48 h, and the colony-forming units were counted. The photosensitizers were investigated using spectroscopy and fluorescence microscopy. There was no significant difference between the culture media (p > 0.05). However, a statistical reduction in both media was observed at 30 min (1058 J/cm2) (p < 0.05). The findings of this study suggest that aBL has the potential to kill bacteria regardless of the culture media used. Light therapy could be a promising and cost-effective strategy for preventing periodontal disease when used in combination with mechanical plaque control.

Effect of Expansion Media on Functional Characteristics of Bone Marrow-Derived Mesenchymal Stromal Cells.

The therapeutic efficacy of mesenchymal stromal cells (MSCs) has been shown to rely on their immunomodulatory and regenerative properties. In order to obtain sufficient numbers of cells for clinical applications, MSCs have to be expanded ex vivo. Expansion media with xenogeneic-free (XF) growth-promoting supplements like human platelet lysate (PL) or serum- and xenogeneic-free (SF/XF) formulations have been established as safe and efficient, and both groups provide different beneficial qualities. In this study, MSCs were expanded in XF or SF/XF media as well as in mixtures thereof. MSCs cultured in these media were analyzed for phenotypic and functional properties. MSC expansion was optimal with SF/XF conditions when PL was present. Metabolic patterns, consumption of growth factors, and secretome of MSCs differed depending on the type and concentration of supplement. The lactate per glucose yield increased along with a higher proportion of PL. Many factors in the supernatant of cultured MSCs showed distinct patterns depending on the supplement (e.g., FGF-2, TGFß, and insulin only in PL-expanded MSC, and leptin, sCD40L PDGF-AA only in SF/XF-expanded MSC). This also resulted in changes in cell characteristics like migratory potential. These findings support current approaches where growth media may be utilized for priming MSCs for specific therapeutic applications.

The effect of amorphous calcium carbonate as a culture media supplement on embryonic development of murine sibling embryos.

PURPOSE: The aim of this study was to compare the addition in culture media of stabilized amorphous calcium carbonate (ACC) versus calcium chloride (CaCl2) or calcium carbonate in crystalline form (CCC) on growth rates among sibling mouse embryos. METHODS: We evaluated the effect of different ACC concentrations on the rates of embryo compaction at 60 h, blastocyst rate at 84 h and percentage of fully hatched at 108 h following hCG injection. As ACC is stabilized by tripolyphosphate (TPP), we also evaluated the addition of TPP alone to the culture media. Finally, we compared supplemented ACC culture media to one-step SAGE and Irvine cleavage media. RESULTS: The results revealed that ACC accelerates the compaction and blastocyst rates, as well as the percentage of fully hatched embryos in a dose-dependent manner, with an increased positive effect at 2.5 mM. The magnitude of the effect for ACC-supplemented media on the embryo developmental rate was between 30 to 40% (p < 0.01) faster for each stage, compared to both SAGE and Irvine one-step standard media. Embryos cultured with SAGE or Irvine media with or without supplementation of CaCl2 or CCC, did not produce the same improvements as observed with ACC. CONCLUSION: In conclusion, the ACC demonstrates a rapid modulation effect for restoring media optimal pH. ACC can inhibit cathepsin B activity during in vitro culture of fibroblast cells. The beneficial impact of ACC on cleavage mouse embryos is likely due to an improved buffering effect causing slower pH media variations, which may enhance quality and implantation potential of embryos following in vitro culture.

Culture Media Supplemented With 10% Equine Serum Provided Chondroprotection in an In Vitro Co-Culture of Cartilage and Synovial Membrane.

No studies have evaluated the effect of culture in serum-free media (SF) vs. media supplemented with equine serum (ES) on co-culture of synovial membrane and cartilage tissue explants. The study objective was to evaluate the effects of equine serum supplementation on induced production of inflammatory and catabolic mediators from articular cartilage and synovial explants while in co-culture. Articular cartilage and synovial membrane explants were harvested from femoropatellar joints of five adult horses. Cartilage and synovial explants were harvested from the stifle of five horses, placed in co-culture, stimulated with IL-1ß (10 ng/ml) and maintained in culture for 3, 6 and 9 days in 10% ES or SF. At each time point, media was harvested for analysis of cellular viability (Lactate dehydrogenase) and elution of glycosaminoglycans (Dimethylene Blue Binding Assay). Tissue explants were harvested for histopathologic and gene expression analyses. No differences in cell viability were observed between SF and ES groups. SF culture produced an upregulation of TNF-α in synovial membrane and ADAMTS-4 and five in articular cartilage at 9 days of culture. ES produced an upregulation of aggrecan expression in cartilage at 9 days of culture. No differences in tissue viability were found between culture media, but SF media produced a higher glycosaminoglycan concentration in media at 3 days of culture. The addition of 10% ES produced a slight chondroprotective effect in an inflamed co-culture system. This effect should be considered when designing studies evaluating treatment of serum or plasma-based orthobiologic studies in vitro.

CYP broth: a tool for Yersinia pestis isolation in ancient culture collections and field samples.

We developed a simple new selective LB-based medium, named CYP broth, suitable for recovering long-term stored Y. pestis subcultures and for isolation of Y. pestis strains from field-caught samples for the Plague surveillance. It aimed to inhibit the growth contaminating microorganisms and enrich Y. pestis growth through iron supplementation. The performance of CYP broth on microbial growth from different gram-negative and gram-positive strains from American Type Culture Collection (ATCC®) and other clinical isolates, field-caught rodent samples, and more importantly, on several vials of ancient Y. pestis subcultures was evaluated. Additionally, other pathogenic Yersinia species such as Y. pseudotuberculosis and Y. enterocolitica were also successfully isolated with CYP broth. Selectivity tests and bacterial growth performance on CYP broth (LB broth supplemented with Cefsulodine, Irgasan, Novobiocin, nystatin and ferrioxamine E) were evaluated in comparison with LB broth without additive; LB broth/CIN, LB broth/nystatin and with traditional agar media including LB agar without additive, and LB agar and Cefsulodin-Irgasan-Novobiocin Agar (CIN agar) supplemented with 50 µg/mL of nystatin. Of note, the CYP broth had a recovery twofold higher than those of the CIN supplemented media or other regular media. Additionally, selectivity tests and bacterial growth performance were also evaluated on CYP broth in the absence of ferrioxamine E. The cultures were incubated at 28 °C and visually inspected for microbiological growth analysis and O.D.625 nm measurement between 0 and 120 h. The presence and purity of Y. pestis growth were confirmed by bacteriophage and multiplex PCR tests. Altogether, CYP broth provides an enhanced growth of Y. pestis at 28 °C, while inhibiting contaminant microorganisms. The media is a simple, but powerful tool to improve the reactivation and decontamination of ancient Y. pestis culture collections and for the isolation of Y. pestis strains for the Plague surveillance from various backgrounds. KEY POINTS: • The newly described CYP broth improves the recuperation of ancient/contaminated Yersinia pestis culture collections • CYP broth was also efficient in reducing environmental contamination in field-capture samples, improving Y. pestis isolation • CYP broth can also be used for the isolation of Y. enterocolitica and Y. pseudotuberculosis.

Amino acid abundance and composition in cell culture medium affects trace metal tolerance and cholesterol synthesis.

Amino acid compositions of cell culture media are empirically designed to enhance cell growth and productivity and vary both across media formulations and over the course of culture due to imbalance in supply and consumption. The interconnected nature of the amino acid transporters and metabolism suggests that changes in amino acid composition can affect cell physiology. In this study, we explore the effect of a step change in amino acid composition from a DMEM: F12-based medium to a formulation varying in relative abundances of all amino acids, evaluated at two amino acid concentrations (lean LAA vs. rich HAA). Cell growth was inhibited in LAA but not HAA. In addition to the expected effects on expression of the cell cycle, amino acid response and mTOR pathway genes in LAA, we observed an unanticipated effect on zinc uptake and efflux genes. This was accompanied by a lower tolerance to zinc supplementation in LAA but not in the other formulations. Histidine was sufficient but not necessary to prevent such zinc toxicity. Additionally, an unanticipated downregulation of genes in the cholesterol synthesis pathway was observed in HAA, accompanied by an increase in cellular cholesterol content, which may depend on the relative abundances of glutamine and other amino acids. This study shows that changes in the amino acid composition without any evident effect on growth may have profound effects on metabolism. Such analyses can help rationalize the designing of medium and feed formulations for bioprocess applications beyond replenishment of consumed components.

Effect of untreated and pretreated sugarcane molasses on growth performance of Haematococcus pluvialis microalgae in inorganic fertilizer and macrophyte extract culture media.

The growth of Haematococcus pluvialis in two alternative culture media NPK (10:10:10) and ME (macrophyte extract), under mixotrophic conditions using sugarcane molasses as a carbon source were evaluated for 28 days. The molasses was used in two different ways, in a native form (untreated) and a hydrolyzed (pretreated). Cell density of Haematococcus pluvialis in mixotrophic cultivation was higher in pretreated molasses. Growth rate was higher when pretreated molasses were employed in mixotrophic cultivation with NPK culture medium (k=0.5 7th growth day). Biomass, chlorophyll-a, conductivity and total inorganic nitrogen were not significantly different (p>0.05) during the experimental period for two mixotrophic cultivation and culture media. Protein contents of H. pluvialis biomass were higher in NPK culture medium with pretreated molasses (50% dry biomass). Annual biomass production was 520 kg-1 dry biomass with untreated molasses for two culture media, and 650 and 520 kg-1 dry biomass with pretreated molasses for NPK and ME culture media, respectively. The use of NPK and ME culture media in mixotrophic cultivation may be a new protocol for H. pluvialis cultivation due to the low cost and similar annual production.

Human Platelet Lysate as Alternative of Fetal Bovine Serum for Enhanced Human In Vitro Bone Resorption and Remodeling.

Introduction: To study human physiological and pathological bone remodeling while addressing the principle of replacement, reduction and refinement of animal experiments (3Rs), human in vitro bone remodeling models are being developed. Despite increasing safety-, scientific-, and ethical concerns, fetal bovine serum (FBS), a nutritional medium supplement, is still routinely used in these models. To comply with the 3Rs and to improve the reproducibility of such in vitro models, xenogeneic-free medium supplements should be investigated. Human platelet lysate (hPL) might be a good alternative as it has been shown to accelerate osteogenic differentiation of mesenchymal stromal cells (MSCs) and improve subsequent mineralization. However, for a human in vitro bone model, hPL should also be able to adequately support osteoclastic differentiation and subsequent bone resorption. In addition, optimizing co-culture medium conditions in mono-cultures might lead to unequal stimulation of co-cultured cells. Methods: We compared supplementation with 10% FBS vs. 10%, 5%, and 2.5% hPL for osteoclast formation and resorption by human monocytes (MCs) in mono-culture and in co-culture with (osteogenically stimulated) human MSCs. Results and Discussion: Supplementation of hPL can lead to a less donor-dependent and more homogeneous osteoclastic differentiation of MCs when compared to supplementation with 10% FBS. In co-cultures, osteoclastic differentiation and resorption in the 10% FBS group was almost completely inhibited by MSCs, while the supplementation with hPL still allowed for resorption, mostly at low concentrations. The addition of hPL to osteogenically stimulated MSC mono- and MC-MSC co-cultures resulted in osteogenic differentiation and bone-like matrix formation, mostly at high concentrations. Conclusion: We conclude that hPL could support both osteoclastic differentiation of human MCs and osteogenic differentiation of human MSCs in mono- and in co-culture, and that this can be balanced by the hPL concentration. Thus, the use of hPL could limit the need for FBS, which is currently commonly accepted for in vitro bone remodeling models.

The amount of membrane cholesterol required for robust cell adhesion and proliferation in serum-free condition.

Serum-containing medium is widely used to support cell attachment, stable growth and serial passaging of various cancer cell lines. However, the presence of cholesterols and lipids in serum greatly hinders the analysis of the effects of cholesterol depletion on cells in culture. In this study, we developed a defined serum-free culture condition accessible to a variety of different types of adherent cancer cells. We tested different factors that are considered essential for cell culture and various extracellular matrix for plate coating, and found cells cultured in Dulbecco's Modified Eagle's Medium (DMEM) basal media supplemented with Albumin (BSA) and insulin-transferrin-selenium-ethanolamine (ITS-X) on fibronectin-precoated plate (called as "DA-X condition") showed comparable proliferation and survival to those in a serum-containing medium. Interestingly, we observed that DA-X condition could be adapted to a wide variety of adherent cancer cell lines, which enabled the analysis of how cholesterol depletion affected cancer cells in culture. Mechanistically, we found the beneficial effects of the DA-X condition in part can be attributed to the appropriate level of membrane cholesterol, and fibronectin-mediated signaling plays an important role in the suppression of cholesterol production.

The crosstalk between endothelial cells and vascular smooth muscle cells aggravates high phosphorus-induced arterial calcification.

Arterial calcification is highly prevalent, particularly in patients with end-stage renal disease (ESRD). The osteogenic differentiation of vascular smooth muscle cells (VSMCs) is the critical process for the development of arterial calcification. However, the detailed mechanism of VSMCs calcification remains to be elucidated. Here, we investigated the role of exosomes (Exos) derived from endothelial cells (ECs) in arterial calcification and its potential mechanisms in ESRD. Accelerated VSMCs calcification was observed when VSMCs were exposed to ECs culture media stimulated by uremic serum or high concentration of inorganic phosphate (3.5 mM Pi). and the pro-calcification effect of the ECs culture media was attenuated by exosome depletion. Exosomes derived from high concentrations of inorganic phosphate-induced ECs (ECsHPi-Exos) could be uptaken by VSMCs and promoted VSMCs calcification. Microarray analysis showed that miR-670-3p was dramatically increased in ECsHPi-Exos compared with exosomes derived from normal concentrations of inorganic phosphate (0.9 mM Pi) induced ECs (ECsNPi-Exos). Mechanistically, insulin-like growth factor 1 (IGF-1) was identified as the downstream target of miR-670-3p in regulating VSMCs calcification. Notably, ECs-specific knock-in of miR-670-3p of the 5/6 nephrectomy with a high-phosphate diet (miR-670-3pEC-KI + NTP) mice that upregulated the level of miR-670-3p in artery tissues and significantly increased artery calcification. Finally, we validated that the level of circulation of plasma exosomal miR-670-3p was much higher in patients with ESRD compared with healthy controls. Elevated levels of plasma exosomal miR-670-3p were associated with a decline in IGF-1 and more severe artery calcification in patients with ESRD. Collectively, these findings suggested that ECs-derived exosomal miR-670-3p could promote arterial calcification by targeting IGF-1, which may serve as a potential therapeutic target for arterial calcification in ESRD patients.

Culture media, DMSO and efflux affect the antibacterial activity of cisplatin and oxaliplatin.

Cisplatin was originally discovered through its antibacterial action and subsequently has found use as a potent broad-spectrum anticancer agent. This study determines the effect of growth media and solvent on the antibacterial activity of cisplatin and its analogue, oxaliplatin. Escherichia coli MG1655 or MG1655 ΔtolC was treated with the platinum compounds under different conditions and susceptibility was determined. Our results showed that DMSO reduced the activity of cisplatin by fourfold (MIC 12·5 mg l-1 ) compared with 0·9% NaCl-solubilized cisplatin (MIC 3·12 mg l-1 ) when tested in MOPS. Surprisingly, complete loss of activity was observed in Mueller-Hinton Broth II (MHB II). By supplementing MOPS with individual components of MHB II such as the sulphur-containing amino acids, l-cysteine and l-methionine, individually or in combination reduced activity by ≥8-fold (MIC ≥25 mg l-1 ). Oxaliplatin was less active against E. coli (MIC 100 mg l-1 ) but exhibited similar inactivation in the presence of DMSO, MHBII or MOPS spiked with l-cysteine and l-methionine (MIC ≥400 mg l-1 ). Our data suggest that the antibacterial activity of cisplatin and oxaliplatin is modulated by both choice of solvent and composition of growth media. We demonstrate that this is primarily due to sulphur-containing amino acids cysteine and methionine, an essential component of the recommended media for testing antimicrobial susceptibility, MHBII.

Lycopene supplementation to serum-free embryo culture medium and its effect on development and quality of bovine blastocysts produced in vitro.

Selenium is commonly used as an antioxidant in a serum-free culture medium setting. However, lycopene has emerged as a potent antioxidant being twice as efficient as ß-carotene and 10 times as efficient as α-tocopherol with beneficial effects when supplemented in a serum-free maturation medium. Here, we aimed to evaluate the effect of lycopene supplementation in a serum-free culture medium on blastocyst development and quality. After in vitro maturation and fertilization, presumed zygotes were cultured in groups of 25 in 50 µl droplets of synthetic oviductal fluid. Culture medium supplementation was done using four experimental groups: insulin, transferrin, selenium (ITS, control); ITS + DMSO (diluent control); ITS + DMSO-lycopene 0.1 µM (ITSL); and IT + DMSO-lycopene 0.1 µM (ITL). DMSO was used as a diluent for lycopene. Blastocyst development among experimental groups was fitted in mixed-effects models, and blastocyst quality parameters (assessed via differential apoptotic staining) were evaluated in mixed linear regression models. The cleavage (85.3 ± 2.4, 82.6 ± 2.7, 86 ± 2.3 and 86.4 ± 2.3% for control, diluent control, ITSL and ITL, respectively) and day 8 blastocyst rates (37.4 ± 3.3, 36.9 ± 3.4, 39.7 ± 3.3 and 46.2 ± 3.4% for control, diluent control, ITSL and ITL, respectively) were not different (p > .1) among experimental groups. Embryos produced in the ITL group resulted in blastocysts with higher total cell numbers (TCN; 141 ± 19.2), inner cell mass (ICM; 65.3 ± 11.6) and trophectoderm cells (TE; 75.2 ± 8.8) compared with the control (129 ± 19.2, 56.3 ± 11.6 and 72.7 ± 8.8, for TCN, ICM and TE; p < .01, respectively). Lycopene-supplemented groups (ITSL and ITL) resulted in blastocysts with similar TCN, ICM and TE (p > .2). The number of apoptotic cells was not different among experimental groups (p > .1). Lycopene supplementation to the culture medium only produced a numerical increase in the blastocyst rate but replacing selenium with lycopene in a serum-free culture medium resulted in blastocysts with more cells.

Supplementation of EGF, IGF-1, and Connexin 37 in IVM Medium Significantly Improved the Maturation of Bovine Oocytes and Vitrification of Their IVF Blastocysts.

The quality and developmental capacity of oocytes derived from in vitro maturation (IVM) remain unsatisfactory, which greatly impairs the efficiency and application of embryo technologies. The present experiment was designed to investigate the effect of the supplementation of EGF, IGF-1, and Cx37 in an IVM medium on the maturation quality and development ability of bovine oocytes. The cytoplasmic maturation events of oocytes and the quality of in vitro fertilization (IVF) blastocysts were examined to investigate the relative mechanisms. Our results showed that the nuclear maturation and blastocyst development after the IVF of oocytes treated with 25 µg/mL Cx37 or the combination of 50 ng/mL EGF and 100 ng/mL IGF-1 were significantly increased compared to those of the control group (p < 0.05). Furthermore, the blastocyst rate, and blastocyst total cell number and survival rate after vitrification of the EGF+IGF-1+Cx37 group, were significantly higher than those of the control group (p < 0.05), but lower than those of the FSH+LH+EGF+IGF-1+Cx37 group (p < 0.05). The transzonal projection (TZP) intensity, glutathione (GSH) level, and mitochondrial function of the EGF+IGF-1+Cx37 group were significantly higher than that of the control group, and lower than those of the FSH+LH+EGF+IGF-1+Cx37 group, in contrast to the results of the reactive oxygen species (ROS) levels. In conclusion, our results showed that the supplementation of 50 ng/mL EGF, 100 ng/mL IGF-1, and 25 µg/mL Cx37 in the IVM of bovine oocytes significantly improved their quality and developmental ability by increasing the TZP, mitochondrial function, and GSH level.

Media Supplementation with Mannitol and Biotin Enhances Squalene Production of Thraustochytrium ATCC 26185 through Increased Glucose Uptake and Antioxidative Mechanisms.

Media supplementation with exogenous chemicals is known to stimulate the accumulation of important lipids produced by microalgae and thraustochytrids. However, the roles of exogenous chemicals in promoting and preserving the terpenoids pool of thraustochytrids have been rarely investigated. Here, we realized the effects of two media supplements-mannitol and biotin-on the biomass and squalene production by a thraustochytrid strain (Thraustochytrium sp. ATCC 26185) and elucidated their mechanism of action. A significant change in the biomass was not evident with the exogenous addition of these supplements. However, with mannitol (1 g/L) supplementation, the ATCC 26185 culture achieved the best concentration (642 ± 13.6 mg/L) and yield (72.9 ± 9.6 mg/g) of squalene, which were 1.5-fold that of the control culture (non-supplemented). Similarly, with biotin supplementation (0.15 mg/L), the culture showed 459 ± 2.9 g/L and 55.7 ± 3.2 mg/g of squalene concentration and yield, respectively. The glucose uptake rate at 24 h of fermentation increased markedly with mannitol (0.31 g/Lh-1) or biotin (0.26 g/Lh-1) supplemented culture compared with non-supplemented culture (0.09 g/Lh-1). In addition, the reactive oxygen species (ROS) level of culture supplemented with mannitol remained alleviated during the entire period of fermentation while it alleviated after 24 h with biotin supplementation. The ∆ROS with mannitol was better compared with biotin supplementation. The total antioxidant capacity (T-AOC) of the supplemented culture was more than 50% during the late stage (72-96 h) of fermentation. Our study provides the potential of mannitol and biotin to enhance squalene yield and the first lines of experimental evidence for their protective role against oxidative stress during the culture of thraustochytrids.

The use of insulin-transferrin-selenium (ITS), and folic acid on individual in vitro embryo culture systems in cattle.

Individual embryo culture is the only strategy that allows the tracking of embryos throughout the culture period. However, this procedure leads to lower embryo development. This study aimed to evaluate different alternatives to improve embryo development in a single in vitro production system. First, embryo production was compared between individual cultures on a 20 µL droplet and Cell-Tak® system. Then, various concentrations of folic acid were tested for use in combination with insulin-transferrin-selenium (ITS). To determine the concentration, embryos were analyzed not only by development but also by their methylation status. Finally, the supplementation of individual culture media with ITS and/or folic acid was evaluated. The results showed that embryos cultured in the Cell-Tak® system presented lower blastocyst rates than the microdroplets system. When the concentration of folic acid was tested, 20 µM and 500 µM presented a higher level of insulin-like growth factor (IGF2) DNA methylation pattern compared to control, suggesting that in vitro conditions alter DNA methylation pattern in that region and folic acid reestablishes the pattern. However, when it was used in an individual culture system, folic acid did not improve embryo development. Conversely, ITS which is composed of three important components, proved to be an alternative to individual embryo culture, improving embryo rates, showing similar rates to grouped culture embryos. Since Folic Acid change epigenetic profile, additional studies are needed to evaluate its use in IVP culture systems.

Influence of glucose on swarming and quorum sensing of Dickeya solani.

Dickeya solani is a pathogen most frequently responsible for infecting potato plants in Europe. As in the case of most plant pathogens, its ability to colonize and invade the host depends on chemotaxis and motility. The coordinated movement of Dickeya over solid surfaces is governed by a quorum sensing mechanism. In D. solani motility is regulated by ExpI-ExpR proteins, homologous to luxI-luxR system from Vibrio fisheri, in which N-acyl-homoserine lactones (AHLs) serve as signaling molecules. Moreover, in many Gram-negative bacteria motility is coupled with central metabolism via carbon catabolite repression. This enables them to reach more nutrient-efficient niches. The aim of this study was to analyze the swarming motility of D. solani depending on the volume of the medium in the cultivation plate and glucose content. We show that the ability of this bacterium to move is strictly dependent on both these factors. Moreover, we analyze the production of AHLs and show that the quorum sensing mechanism in D. solani is also influenced by the availability of glucose in the medium and that the distribution of these signaling molecules are different depending on the volume of the medium in the plate.

Supplementation of freezing medium with encapsulated or free glutathione during cryopreservation of bull sperm.

The objective was to compare effects of encapsulated or free glutathione (GSH) on the quality of frozen-thawed bull sperm. Ejaculates were collected via artificial vagina from six mature Holstein bulls once weekly for 6 weeks. All ejaculates had motility ≥70%, sperm concentration ≥1.0 × 109 /ml and ≤15% morphologically abnormal sperm. Each week, semen was pooled and diluted with lecithin-based extenders containing various concentrations of encapsulated (E0, E1, E2.5 and E5 mM) or free (F0, F1, F2.5 and F5 mM) GSH, with total glutathione content determined before and after cryopreservation. Total GSH in fresh semen was (mean+SEM) 4.8 ± 0.2 nmol/108 sperm, whereas in frozen-thawed semen of group F0 (control), it decreased to 1.4 ± 0.2 nmol/108 sperm, a 70.8% reduction (p < .05). In addition, total GSH in frozen-thawed semen from groups E2.5, E5 and F5 were 2.4 ± 0.2, 2.8 ± 0.2 and 1.8 ± 0.2 nmol/108 sperm, respectively (E5 versus. F0, p < .05). Compared to group F0, frozen-thawed sperm from group E2.5 had greater (p < .05) percentages of sperm that were viable (Annexin-V) (61.1 ± 1.8 versus. 71.1 ± 1.8) and that had cell membrane integrity (eosin-nigrosin) (64.5 ± 3.1 versus. 80.0 ± 3.1). Furthermore, frozen-thawed sperm from group E2.5 had the numerically highest total and progressive motility (CASA) and cell membrane functionality (HOS) and the lowest percentage of early apoptotic sperm (Annexin-V). However, acrosome membrane integrity (PSA) of E5 had the lowest mean (p < .05), whereas E2.5 caused a small nonsignificant decrease (69.1 ± 1.4%) compared to E0 and F0. In conclusion, 2.5 mM encapsulated GSH in semen extender significantly improved the quality of frozen-thawed bull sperm.

Effect of Different Zinc Sources and Concentrations on the Biomass Yield of Saccharomyces cerevisiae Yeast.

Zinc incorporation and the growth of Saccharomyces cerevisiae were investigated in a culture supplemented with three inorganic zinc salts, i.e. zinc sulphate, zinc chloride and zinc nitrate. The cultivation was performed on a yeast extract peptone dextrose (YEPD) broth medium. The growth of yeast was carried out at different concentrations of zinc, i.e. 0, 30, 60, 90 and 120 mg 100 ml-1. It was found that the addition of different zinc sources at 30 mg 100 ml-1 concentration produced higher biomass yield ranging 1.00-1.03 g from 100 ml-1 of cultivation medium, while higher zinc concentration in the medium caused significantly lower yields of yeast biomass. The amount of zinc in yeast cells was determined by an atomic absorption spectrometer (AAS). The highest amount of zinc in yeast cells was achieved when added in the form of zinc sulphate at a concentration of 120 mg 100 ml-1. The increment of intracellular zinc was up to 9889.67 mg kg-1 of biomass.