Chemical gasification: An alternative approach to in vitro maturation of bovine oocytes.

This study aimed to evaluate the effect of chemical gasification and HEPES as alternative systems to pH control during in vitro maturation on bovine oocytes competence. Groups of 20 bovine cumulus oocytes complexes (COCs) were randomly distributed and cultured for 24 h in one of the following experimental groups: (i) chemical reaction (ChRG) system: CO2 generated from sodium bicarbonate and citric acid reaction (ii) culture media TCM-HEPES (HEPES-G); and (iii) control group (CNTG) in conventional incubator. After in vitro maturation (IVM), the COCs were in vitro fertilized (IVF), and in vitro cultivated (IVC) in a conventional incubator. We evaluated oocyte nuclear maturation, cleavage and blastocyst rates, in addition to the relative mRNA expression of BAX, BMP-15, AREG and EREG genes in oocytes and cumulus cells. The proportion of oocytes in metaphase II was higher in CNTG and ChRG (77.57% and 77.06%) than in the HEPES-G (65.32%; p = .0408 and .0492, respectively). The blastocyst production was similar between CNTG and ChRG (26.20% and 28.47%; p = .4232) and lower (p = .001) in the HEPES-G (18.71%). The relative mRNA expression of BAX gene in cumulus cells was significantly higher (p = .0190) in the HEPES-G compared to the CNTG. Additionally, the relative mRNA expression of BMP-15 gene was lower (p = .03) in oocytes from HEPES-G compared to the CNTG. In conclusion, inadequate atmosphere control has a detrimental effect on oocyte maturation. Yet, the use of chemical gasification can be an efficient alternative to bovine COCs cultivation.

A dye elution method for the quantification of insecticidal crystal proteins from Bacillus thuringiensis and its compatibility with the presence of agro-industrial raw materials and waste products.

The insecticidal crystal proteins produced by Bacillus thuringiensis during sporulation are active ingredients against lepidopteran, dipteran, and coleopteran insects. Several methods have been reported for their quantification, such as crystal counting, ELISA, and SDS-PAGE/densitometry. One of the major tasks in industrial processes is the analysis of raw material dependency and costs. Thus, the crystal protein quantification method is expected to be compatible with the presence of complex and inexpensive culture medium components. This work presents a revalidated elution-based method for the quantification of insecticidal crystal proteins produced by the native strain B. thuringiensis RT. To quantify proteins, a calibration curve was generated by varying the amount of BSA loaded into SDS-PAGE gels. First, SDS-PAGE was performed for quality control of the bioinsecticide. Then, the stained protein band was excised from 10% polyacrylamide gel and the protein-associated dye was eluted with an alcoholic solution of SDS (3% SDS in 50% isopropanol) during 45 min at 95°C. This protocol was a sensitive procedure to quantify proteins in the range of 2.0-10.0 µg. As proof of concept, proteins of samples obtained from a complex fermented broth were separated by SDS-PAGE. Then, Cry1 and Cry2 proteins were properly quantified.

Comparison of techniques for corneal epithelium cell culture for the collection of conditioned medium / Comparação das técnicas de cultivo de células epiteliais da córnea para a coleta de meio condicionado

ABSTRACT Purposes: To determine the best protocol in obtaining the higher yield of conditioned culture medium to be used for the bone marrow mesenchymal stem cell differentiation into corneal epithelial cells, five techniques for the primary culture of human corneal epithelial cells were evaluated. Methods: The studied culture techniques of corneal epithelial cells were: explants in culture flasks with and without hydrophilic surface treatment, on amniotic membrane, with enzymatic digestion, and by corneal scraping. The conditioned culture medium collected from these cultures was used to differentiate human bone marrow mesenchymal stem cells into corneal epithelial cells, which were characterized using flow cytometry with pan-cytokeratin and the corneal-specific markers, cytokeratin 3 and cytokeratin 12. Results: The culture technique using flasks with hydrophilic surface treatment resulted in the highest yield of conditioned culture medium. Flasks without surface treatment resulted to a very low success rate. Enzymatic digestion and corneal scraping showed contamination with corneal fibroblasts. The culture on amniotic membranes only allowed the collection of culture medium during the 1st cell confluence. The effectiveness of cell differentiation was confirmed by cytometry analysis using the collected conditioned culture medium, as demonstrated by the expressions of cytokeratin 3 (95.3%), cytokeratin 12 (93.4%), and pan-cytokeratin (95.3%). Conclusion: The culture of corneal epithelial cell explants in flasks with hydrophilic surface treatment is the best technique for collecting a higher yield of conditioned culture medium to be used to differentiate mesenchymal stem cells.

RESUMO Objetivos: Foram estudadas cinco técnicas de cultivo primário de células epiteliais de córnea humana para se determinar o melhor protocolo para a obtenção do maior rendimento de meio de cultivo condicionado para ser utilizado na diferenciação de células tronco mesenquimais para células epiteliais de córnea. Métodos: As técnicas de cultivo estudadas foram: explantes em frascos de cultivo com e sem tratamento hidrofílico de superfície, sobre membrana amniótica, com digestão enzimática e por raspado de córnea. O meio de cultivo condicionado foi coletado e as células tronco mesenquimais induzidas a se diferenciarem em células epiteliais da córnea utilizando o meio de cultivo condicionado. As células foram caracterizadas por citometria de fluxo com pan-citoqueratina e com os marcadores específicos da córnea, citoqueratina 3 e citoqueratina 12. Resultados: A técnica utilizando frascos com o tratamento de superfície apresentou o maior rendimento de meio de cultivo condicionado. Os frascos sem tratamento de superfície levaram a uma taxa de sucesso muito baixa. A digestão enzimática e a raspagem da córnea mostraram contaminação das culturas com fibroblastos de córnea. A cultura sobre membranas amnióticas só permitiu a coleta do meio de cultivo condicionado durante a 1ª confluência celular. A análise de citometria de fluxo confirmou o sucesso da diferenciação celular utilizando o meio de cultivo condicionado coletado, demonstrada pela expressão de citoqueratina 3 (95,3%), citoqueratina 12 (93,4%) e pan-citoqueratina (95,3%). Conclusão: O cultivo de explantes de células tronco mesenquimais em frascos com tratamento hidrofílico de superfície é a melhor técnica para a obtenção de um alto rendimento de meio de cultivo condicionado.

Low-cost culture media designed for biomass production of beneficial lactic acid bacteria for their inclusion in a formula to treat bovine reproductive infections.

As a first step for the use of probiotics in a formula for cattle, it is required to have available low-cost culture medium(s) and efficient production conditions for the growth of probiotic bacteria and high production of cell biomass. De Man-Rogosa-Sharpe medium, used frequently for lactic acid bacteria (LAB) contains adequate ingredients for their growth but is very expensive for industrial application. The nutrients required for LAB growth are strain-dependent. In this work, traditional culture media were evaluated omitting and/or modifying ingredients in their composition, as carbon or nitrogen source, on the basis of their low-cost industrial waste, to select those supporting the most efficient growth. The results showed that the formulation of culture media containing fructose (0.5%) and molasses (1.0%) was better for the growth and production of cell biomass for all the strains assayed, except Lactobacillus gasseri CRL1421 growing in 1.5% corn syrup. FM902 yeast extract at concentrations between 1.5% and 2.5% was the most adequate for most of the strains. The LAB grown in the designed media maintained the beneficial properties for which they selected. The use of the culture media designed to produce biomass decrease production costs, which is an important step for the feasible industrial production of probiotic pharmaceuticals.

How to optimize culture media osmolality during Assisted Reproductive Technologies treatments.

OBJECTIVE: The objective of our study was to compare the osmolality in sequential and single step culture media, used for in vitro human embryo culture, covered with mineral oil and paraffin, in dry and humid incubators. METHODS: We performed a prospective observational study. A total of 120 Petri dishes, with 960 droplets of culture media, were evaluated. Each dish was prepared with 4 droplets of single step medium and sequential medium. Sixty dishes were covered with mineral oil and 60 with paraffin oil. Half were incubated in a dry incubator and half in a humid. Osmolality was measured on days 1, 3, 5, 7. ANOVA test was performed for statistical analysis. RESULTS: Osmolality results for single step and sequential medium, that were covered with both mineral and paraffin oil and placed in the dry incubator, significantly increased throughout the study time (D7>D5>D3). In the humid incubator, the results were similar for all periods. Osmolality was significantly lower in humid incubator, in all periods, when droplets were covered with both oils. When both culture media were placed in the humid incubator, no variation was detected, using both oils. However, when single step medium was placed in the dry incubator, covered with mineral oil, we observed a higher osmolality than the covered with paraffin oil. CONCLUSIONS: TWe can conclude that humid incubator is better for maintaining osmolality and paraffin oil protect single step media from evaporation in dry incubator.

Hyaluronic acid production and purification techniques: a review.

Hyaluronic acid (HA) is an exopolysaccharide extracted from several sources such as rooster combs, umbilical cords and microorganisms. A system that controls temperature, agitation and aeration of bacterial cultures could make the HA production autonomous. Therefore, HA of microbial origin is set to take over alternative methods of production. Furthermore, the use of different nutrient sources in the culture medium and the purification stage applied in the process can cause physicochemical alterations on the bioproduct. For instance, structural modifications that change the molecular weight of HA may alter its elastic and viscoelastic properties. As a result, HA synthesized by microbes has applications in pharmacology, biotechnology, and tissue engineering. Our aim here, is to show the vast range of applications by compiling articles and patents on the culture media or genetic modifications of microorganisms that synthesize HA.

Evaluation of Conditions to Improve Biomass Production by Submerged Culture of Ganoderma sp.

In the present investigation, the conditions for in vitro submerged culture of a native strain of Ganoderma sp. were evaluated. Different culture medium ingredients, inoculum concentrations, inoculation methods, configuration, and airflows were evaluated to improve biomass production. The addition of thiamine and olive oil to the culture medium increased biomass production, as well as inoculating 6.6 g/L since there are no significant differences in biomass growth according to inoculum origin (pre-inoculum, discs or with spores). The best configuration of the 3 L stirred tank bioreactor was using three impellers and a porous air diffuser of 0.25 volume per volume per minute (vvm), the dry biomass concentration was 22.6 g/L after 12 days of cultivation at 30 °C, much higher than other investigations. This study provides relevant information for pilot-scale production of this fungus for future secondary metabolites. The culture medium was optimized, and it was defined that the concentration and origin of the inoculum did not influence the growth of Biomass, but the aeration and the configuration of the system allowed the establishment of protocols for the cultivation of Ganoderma sp.

Study of protease activity from Aspergillus awamori INCQS2B.361U2/1 extracellular fraction and modification of culture medium composition to isolate a novel aspartic protease.

Aspergillus awamori was cultivated in a modified Breccia medium, and the extracellular fraction was obtained, which presented 260 ± 15 µg of protein/mg and specific protease activity of 3.87 ± 0.52 mM.min-1.mg of protein-1 using Nα-p-tosyl-L-arginine methyl ester hydrochloride (L-TAME) as substrate. This fraction showed major proteins about 104 and 44 kDa and maximal protease activity at pH 5.5, 6.5, and 9.0, suggesting that A. awamori secretes acidic, neutral, and alkaline proteases with expressive thermal stability, however, aspartic protease was the most important activity. When yeast extract was supplemented to a modified Breccia medium, A. awamori protein secretion and protease activity were maximal and the affinity chromatography on pepstatin-agarose was employed to isolate the aspartic protease activity, which was called ASPA, with approximately 75 kDa. ASPA maximal activity was obtained at pH 4.5 and 6.5, and 50 °C. Pepstatin inhibited about 80% of ASPA activity, with IC50 and Ki values of 0.154 and 0.072 µM, respectively. ASPA cleaved protein and peptides substrates with the highest activity against gelatin (95 U/mg) and good peptidase activity with KM 0.0589 mM and Vmax 1.909 mM.min-1.mg protein-1, using L-TAME as substrate. A. awamori extracellular fraction is a source of proteases with important activity, and the supplementation of modified Breccia medium increased the aspartic protease production. This enzyme presented different biochemical characteristics from the previously reported A. awamori aspartic proteases. Therefore, ASPA is an excellent candidate for biotechnological application due to its important activity and thermostability.

Growth dynamic of Leptospira spp. from Sejroe serogroup in different media formulae.

The culturing of Leptospira strains from bovine clinical samples is challenging and has resulted in some gaps in securing an epidemiological understanding. Strains related to chronic reproductive leptospirosis in cattle belong to the Sejroe serogroup - not only Hardjoprajitno and Hardjobovis but also Guaricura genotypes. This study analyses the growth of Leptospira strains from serogroup Sejroe in different culture media, with the aim of suggesting better culturing approaches. To meet this objective, two culture media were applied: EMJH and T80/40/LH. In addition, three different cocktails of selective agents were chosen. The combinations of medium and selective additives resulted in 10 different tested formulae. The poor performance of Hardjobovis in EMJH indicated that its growth may represent a possible bias when culturing these strains from bovine samples. The most efficient medium for culturing Hardjobovis was T80/40/LH, while T80/40/LH medium + STAFF combination proved to be the best choice for growth, being recommended for obtaining a higher number of these strains from bovines.

In vitro maturation medium supplementation: utilization of repaglinide, L-carnitine, and mesenchymal stem cell-conditioned medium to improve developmental competence of oocytes derived from endometriosis mouse models

Endometriosis (EMS) is one of the most prevalent causes for female infertility. Herein, we investigated the effect of the repaglinide (RG), L-carnitine (LC), and bone marrow mesenchymal stem cell-conditioned medium (BMSC-CM) supplementation during in vitro maturation (IVM) on the quality, maturation, and fertilization rates, as well as embryonic quality and development of oocytes derived from normal and EMS mouse model. Immature oocytes were collected from two groups of normal and EMS-induced female NMRI mice at 6-8 weeks of age. Oocytes were cultured in IVM medium unsupplemented (control group), or supplemented with 1 M RG, 0.3 and 0.6 mg/mL LC, and 25 and 50% BMSC-CM. After 24 h of oocyte incubation, IVM rate and antioxidant status were assessed. Subsequently, the rates of fertilization, cleavage, blastulation, and embryonic development were assessed. Our results demonstrated that supplementation of IVM medium with LC and BMSC-CM, especially 50% BMSC-CM, significantly enhanced IVM and fertilization rates, and markedly improved blastocyst development and total blastocyst cell numbers in EMS-induced mice compared to the control group (53.28±0.24 vs 18.09±0.10%). Additionally, LC and BMSC-CM were able to significantly modulate EMS-induced nitro-oxidative stress by boosting total antioxidant capacity (TAC) and mitigating nitric oxide (NO) levels. Collectively, LC and BMSC-CM supplementation improved oocyte quality and IVM rates, pre-implantation developmental competence of oocytes after in vitro fertilization, and enhanced total blastocyst cell numbers probably by attenuating nitro-oxidative stress and accelerating nuclear maturation of oocytes. These outcomes may provide novel approaches to refining the IVM conditions that can advance the efficiency of assisted reproductive technologies in infertile couples.

Effectiveness of near-infrared spectroscopy as a non-invasive tool to discriminate spectral profiles of in vitro cultured oocytes from goats.

Here, we aimed to discriminate between the spectral profiles of spent culture media after oocyte in vitro maturation (IVM) and culture (IVC) from goats of different ages subjected to repeated hormonal treatments. The profiles were discriminated using near infrared (NIR) spectroscopy combined with multivariate methods. A total of 19 goats (young = 10; old = 9) were subjected to serial hormonal stimulation (HS) with gonadotropins. Cumulus oophorus complexes (COCs) were collected using laparoscopic ovum pick-up (LOPU) and subjected to IVM and parthenogenetic activation. The initial embryos were subjected to IVC. Spent culture media were collected after oocyte IVM and on day 2 of IVC and analyzed using NIR spectroscopy. NIR spectral data were interpreted through chemometric methods, such as principle component analysis (PCA) and partial least square discriminant analysis (PLS-DA). The results of PCA analysis clearly showed a separation in the spectral profiles between the experimental groups (HS sessions; young and old animals) both after IVM and IVC. Overall, the main absorption bands were attributed to the C-H group second overtone, first overtone of O-H and N-H, and C-H combinations and may serve as molecular markers. On the other hand, the spectral data obtained using PLS-DA models provided a better classification of the groups. The results showed the possibility of discriminating young and old groups as well as the three HS sessions with high specificity, sensitivity, and accuracy using NIR spectra. Thus, the culture medium analysis using NIR spectroscopy combined with multivariate methods indicated the dissimilarities between the groups and provided an insight into the in vitro development of goat oocytes. This technique serves as an efficient, objective, rapid, and non-invasive method to discriminate spectral profiles.

Evaluación del cultivo in vitro de una cepa mexicana de Ganoderma lucidum / Evaluation of in vitro cultivation of a Mexican strain of Ganoderma lucidum

RESUMEN La cepa mexicana CP-145 de Ganoderma lucidum debido a la importancia medicinal que ha presentado últimamente, la presente investigación tuvo como objetivo evaluar el efecto de la temperatura y medio de cultivo sobre el crecimiento micelial óptimo en diferentes rangos de pH. Los tratamientos correspondieron en la utilización del medio de cultivo papa dextrosa agar (PDA) y extracto de malta agar (EMA), con dos niveles de temperatura (25 y 28 °C) y seis rangos de pH (4.0, 4.5, 5.0, 5.5, 6.0 y 6.5). El diseño experimental fue completamente al azar con medidas repetidas a través del tiempo, analizados con el paquete REPEATED MEASURE y el efecto tiempo con PROC MIXED de SAS. Como resultado se obtuvieron que el efecto de la temperatura y medios de cultivo en los diferentes rangos de pH, presentaron diferencias significativas (P ≤ 0.05). El crecimiento micelial óptimo de la cepa mexicana de G. lucidum fue en el medio de cultivo EMA en los rangos de pH de 4.0 y 4.5 con 8.3 y 8.2 cm respectivamente. De igual forma, en los rangos de pH 4.0 y 4.5 se obtuvieron los crecimientos miceliales óptimos a temperatura de 25 °C con 8.1 y 8.0 cm respectivamente. El cual concluyó esta investigación que el crecimiento micelial óptimo de la cepa mexicana fueron a pH 4.0 y 4.5, temperatura de 25 °C y medio de cultivo EMA.

ABSTRACT The Mexican strain CP-145 of Ganoderma lucidum due to the medicinal importance it has presented lately, the present investigation had as objective to evaluate the effect of temperature and culture medium on the optimal mycelial growth in different pH ranges. The treatments corresponded to the use of potato dextrose agar (PDA) and malt extract agar (EMA), with two temperature levels (25 and 28 °C) and six pH ranges (4.0, 4.5, 5.0, 5.5, 5.5, 6.0 and 6.5). The experimental design was completely randomised with repeated measures over time, analysed with the REPEATED MEASURE package and the time effect with PROC MIXED of SAS. As a result, the effect of temperature and culture media in the different pH ranges showed significant differences (P ≤ 0.05). The optimal mycelial growth of the Mexican strain of G. lucidum was in the EMA culture medium in the pH ranges of 4.0 and 4.5 with 8.3 and 8.2 cm respectively. Similarly, in the pH ranges 4.0 and 4.5 the optimum mycelial growth was obtained at 25 °C with 8.1 and 8.0 cm respectively. This research concluded that the optimal mycelial growth of the Mexican strain was at pH 4.0 and 4.5, temperature of 25 °C and EMA culture medium.

Culture media based on effluent derived from soy protein concentrate production for Lacticaseibacillus paracasei 90 biomass production: statistical optimisation, mineral characterization, and metabolic activities.

The waste and by-products of the soybean industry could be an economic source of nutrients to satisfy the high nutritional demands for the cultivation of lactic acid bacteria. The aims of this work were to maximize the biomass production of Lacticaseibacillus paracasei 90 (L90) in three culture media formulated from an effluent derived from soy protein concentrate production and to assess the effects these media have on the enzymatic activity of L90, together with their influence on its fermentation profile in milk. The presence of essential minerals and fermentable carbohydrates (sucrose, raffinose, and stachyose) in the effluent was verified. L90 reached high levels of microbiological counts (∼ 9 log cfu mL-1) and dry weight (> 1 g L-1) on the three optimized media. Enzymatic activities (lactate dehydrogenase and ß-galactosidase) of L90, and its metabolism of lactose and citric acid, as well as lactic acid and pyruvic acid production in milk, were modified depending on the growth media. The ability of the L90 to produce the key flavour compounds (diacetyl and acetoin) was maintained or improved by growing in the optimized media in comparison with MRS.

Nanoparticles from culture media are internalized by in vitro-produced bovine embryos and its depletion affect expression of pluripotency genes.

Extracellular vesicles are nanoparticles secreted by cell and have been proposed as suitable markers to identify competent embryos produced in vitro. Characterizing EVs secreted by individual embryos is challenging because culture medium itself contributes to the pool of nanoparticles that are co-isolated. To avoid this, culture medium must be depleted of nanoparticles that are present in natural protein source. The aim of this study was to evaluate if the culture medium subjected to nanoparticle depletion can support the proper in vitro development of bovine embryos. Zygotes were cultured in groups on depleted or control medium for 8 days. Nanoparticles from the medium were characterized by their morphology, size and expression of EVs surface markers. Isolated nanoparticles were labelled and added to depleted medium containing embryos at different developmental stages and evaluated after 24 hours at 2, 8-16 cells, morula and blastocyst stages. There were no statistical differences on blastocyst rate at day 7 and 8, total cell count neither blastocyst diameter between groups. However, morphological quality was better in blastocysts cultured in non-depleted medium and the expression of SOX2 was significantly lower whereas NANOG expression was significantly higher. Few nanoparticles from medium had a typical morphology of EVs but were positive to specific surface markers. Punctuated green fluorescence near the nuclei of embryonic cells was observed in embryos from all developmental stages. In summary, nanoparticles from culture medium are internalized by in vitro cultured bovine embryos and their depletion affects the capacity of medium to support the proper embryo development.

A Chemically Defined, Xeno- and Blood-Free Culture Medium Sustains Increased Production of Small Extracellular Vesicles From Mesenchymal Stem Cells.

Cell therapy is witnessing a notable shift toward cell-free treatments based on paracrine factors, in particular, towards small extracellular vesicles (sEV), that mimic the functional effect of the parental cells. While numerous sEV-based applications are currently in advanced preclinical stages, their promised translation depends on overcoming the manufacturing hurdles posed by the large-scale production of purified sEV. Unquestionably, the culture medium used with the parental cells plays a key role in the sEV's secretion rate and content. An essential requisite is the use of a serum-, xeno-, and blood-free medium to meet the regulatory entity requirements of clinical-grade sEV's production. Here, we evaluated OxiumTMEXO, a regulatory complying medium, with respect to production capacity and conservation of the EV's characteristics and functionality and the parental cell's phenotype and viability. A comparative study was established with standard DMEM and a commercially available culture medium developed specifically for sEV production. Under similar conditions, OxiumTMEXO displayed a three-fold increase of sEV secretion, with an enrichment of particles ranging between 51 and 200 nm. These results were obtained through direct quantification from the conditioned medium to avoid the isolation method's interference and variability and were compared to the two culture media under evaluation. The higher yield obtained was consistent with several harvest time points (2, 4, and 6 days) and different cell sources, incluiding umbilical cord-, menstrual blood-derived mesenchymal stromal cells and fibroblasts. Additionally, the stem cell phenotype and viability of the parental cell remained unchanged. Furthermore, OxiumTMEXO-sEV showed a similar expression pattern of the vesicular markers CD63, CD9, and CD81, with respect to sEV derived from the other conditions. The in vitro internalization assays in different target cell types and the pharmacokinetic profile of intraperitoneally administered sEV in vivo indicated that the higher EV production rate did not affect the uptake kinetics or the systemic biodistribution in healthy mice. In conclusion, the OxiumTMEXO medium sustains an efficient and robust production of large quantities of sEV, conserving the classic functional properties of internalization into acceptor target cells and biodistribution in vivo, supplying the amount and quality of EVs for the development of cell-free therapies.

Improvement in extracellular secretion of recombinant L-asparaginase II by Escherichia coli BL21 (DE3) using glycine and n-dodecane.

L-asparaginase II (ASNase) is the biopharmaceutical of choice for the treatment of acute lymphoblastic leukaemia. In this study, E. coli BL21 (DE3) transformed with the pET15b + asnB vector which expresses recombinant ASNase was used as a source to obtain this enzyme. The ideal conditions to produce ASNase would be a high level of secretion into the extracellular medium, which depends not only on the application of molecular biology techniques but also on the development of a strategy to modify cell permeability such as the addition of substances to the culture medium that stimulate destabilisation of structural components of the cell. Thus, the growth of E. coli BL21 (DE3) in modified Luria-Bertani broth, supplemented with 0.8% (w/v) glycine and 6% (v/v) n-dodecane, increased the total yield of ASNase by about 50% (15,108 IU L-1) and resulted in a 16-fold increase in extracellular enzymatic productivity (484 IU L-1 h-1), compared to production using the same medium without addition of these substances. Most of the enzyme (89%) was secreted into the culture medium 24 h after the induction step. This proposed approach presents a simple strategy to increase extracellular production of ASNase in E. coli.

Vinasse: from a residue to a high added value biopolymer.

This work aimed to study the feasibility of using vinasse for polyhydroxybutyrate (PHB) production by Bacillus megaterium. To optimize the culture medium, a Box-Behnken design was employed considering carbon (C), nitrogen (N), and phosphorus (Ph) concentrations as independent variables and PHB productivity as the response variable. The productivity decreased when C or N were increased, probably due to the presence of phenolic compounds and the limitation of N for the production of PHB by Bacillus sp. bacteria. An additional experimental design to optimize the C/N ratio and growing conditions (fermentation time and temperature) was carried out. Fermentation time had a statistically significant effect on PHB productivity reaching 10.6 mg/L h. On the other hand, the variability in physicochemical properties of vinasse samples led to significant differences in PHB productivity. Lower productivity values were obtained when vinasse had higher values of DBO. Therefore, biopolymers production from vinasse is a feasible alternative to valorize this bioethanol by-product.

Nanoparticles from culture media are internalized by in vitro-produced bovine embryos and its depletion affect expression of pluripotency genes

Abstract Extracellular vesicles are nanoparticles secreted by cell and have been proposed as suitable markers to identify competent embryos produced in vitro. Characterizing EVs secreted by individual embryos is challenging because culture medium itself contributes to the pool of nanoparticles that are co-isolated. To avoid this, culture medium must be depleted of nanoparticles that are present in natural protein source. The aim of this study was to evaluate if the culture medium subjected to nanoparticle depletion can support the proper in vitro development of bovine embryos. Zygotes were cultured in groups on depleted or control medium for 8 days. Nanoparticles from the medium were characterized by their morphology, size and expression of EVs surface markers. Isolated nanoparticles were labelled and added to depleted medium containing embryos at different developmental stages and evaluated after 24 hours at 2, 8-16 cells, morula and blastocyst stages. There were no statistical differences on blastocyst rate at day 7 and 8, total cell count neither blastocyst diameter between groups. However, morphological quality was better in blastocysts cultured in non-depleted medium and the expression of SOX2 was significantly lower whereas NANOG expression was significantly higher. Few nanoparticles from medium had a typical morphology of EVs but were positive to specific surface markers. Punctuated green fluorescence near the nuclei of embryonic cells was observed in embryos from all developmental stages. In summary, nanoparticles from culture medium are internalized by in vitro cultured bovine embryos and their depletion affects the capacity of medium to support the proper embryo development.

Effectiveness of near-infrared spectroscopy as a non-invasive tool to discriminate spectral profiles of in vitro cultured oocytes from goats

Abstract Here, we aimed to discriminate between the spectral profiles of spent culture media after oocyte in vitro maturation (IVM) and culture (IVC) from goats of different ages subjected to repeated hormonal treatments. The profiles were discriminated using near infrared (NIR) spectroscopy combined with multivariate methods. A total of 19 goats (young = 10; old = 9) were subjected to serial hormonal stimulation (HS) with gonadotropins. Cumulus oophorus complexes (COCs) were collected using laparoscopic ovum pick-up (LOPU) and subjected to IVM and parthenogenetic activation. The initial embryos were subjected to IVC. Spent culture media were collected after oocyte IVM and on day 2 of IVC and analyzed using NIR spectroscopy. NIR spectral data were interpreted through chemometric methods, such as principle component analysis (PCA) and partial least square discriminant analysis (PLS-DA). The results of PCA analysis clearly showed a separation in the spectral profiles between the experimental groups (HS sessions; young and old animals) both after IVM and IVC. Overall, the main absorption bands were attributed to the C-H group second overtone, first overtone of O-H and N-H, and C-H combinations and may serve as molecular markers. On the other hand, the spectral data obtained using PLS-DA models provided a better classification of the groups. The results showed the possibility of discriminating young and old groups as well as the three HS sessions with high specificity, sensitivity, and accuracy using NIR spectra. Thus, the culture medium analysis using NIR spectroscopy combined with multivariate methods indicated the dissimilarities between the groups and provided an insight into the in vitro development of goat oocytes. This technique serves as an efficient, objective, rapid, and non-invasive method to discriminate spectral profiles.

Effectiveness of near-infrared spectroscopy as a non-invasive tool to discriminate spectral profiles of in vitro cultured oocytes from goats

Here, we aimed to discriminate between the spectral profiles of spent culture media after oocyte in vitro maturation (IVM) and culture (IVC) from goats of different ages subjected to repeated hormonal treatments. The profiles were discriminated using near infrared (NIR) spectroscopy combined with multivariate methods. A total of 19 goats (young = 10; old = 9) were subjected to serial hormonal stimulation (HS) with gonadotropins. Cumulus oophorus complexes (COCs) were collected using laparoscopic ovum pick-up (LOPU) and subjected to IVM and parthenogenetic activation. The initial embryos were subjected to IVC. Spent culture media were collected after oocyte IVM and on day 2 of IVC and analyzed using NIR spectroscopy. NIR spectral data were interpreted through chemometric methods, such as principle component analysis (PCA) and partial least square discriminant analysis (PLS-DA). The results of PCA analysis clearly showed a separation in the spectral profiles between the experimental groups (HS sessions; young and old animals) both after IVM and IVC. Overall, the main absorption bands were attributed to the C-H group second overtone, first overtone of O-H and N-H, and C-H combinations and may serve as molecular markers. On the other hand, the spectral data obtained using PLS-DA models provided a better classification of the groups. The results showed the possibility of discriminating young and old groups as well as the three HS sessions with high specificity, sensitivity, and accuracy using NIR spectra. Thus, the culture medium analysis using NIR spectroscopy combined with multivariate methods indicated the dissimilarities between the groups and provided an insight into the in vitro development of goat oocytes. This technique serves as an efficient, objective, rapid, and non-invasive method to discriminate spectral profiles.(AU)