Scalable in vitro production of defined mouse erythroblasts

Mouse embryonic stem cells (mESCs) can be manipulated in vitro to recapitulate the process of erythropoiesis, during which multipotent cells undergo lineage specification, differentiation and maturation to produce erythroid cells. Although useful for identifying specific progenitors and precursors, this system has not been fully exploited as a source of cells to analyse erythropoiesis. Here, we establish a protocol in which characterised erythroblasts can be isolated in a scalable manner from differentiated embryoid bodies (EBs). Using transcriptional and epigenetic analysis, we demonstrate that this system faithfully recapitulates normal primitive erythropoiesis and fully reproduces the effects of natural and engineered mutations seen in primary cells obtained from mouse models. We anticipate this system to be of great value in reducing the time and costs of generating and maintaining mouse lines in a number of research scenarios.

The Interactions among Isolates of Lactiplantibacillus plantarum and Dairy Yeast Contaminants: Towards Biocontrol Applications

Yeast diversity in the cheese manufacturing process and in the cheeses themselves includes indispensable species for the production of specific cheeses and undesired species that cause cheese defects and spoilage. The control of yeast contaminants is problematic due to limitations in sanitation methods and chemicals used in the food industry. The utilisation of lactic acid bacteria and their antifungal products is intensively studied. Lactiplantibacillus plantarum is one of the most frequently studied species producing a wide spectrum of bioactive by-products. In the present study, twenty strains of L. plantarum from four sources were tested against 25 species of yeast isolated from cheeses, brines, and dairy environments. The functional traits of L. plantarum strains, such as the presence of class 2a bacteriocin and chitinase genes and in vitro production of organic acids, were evaluated. The extracellular production of bioactive peptides and proteins was tested using proteomic methods. Antifungal activity against yeast was screened using in vitro tests. Testing of antifungal activity on artificial media and reconstituted milk showed significant variability within the strains of L. plantarum and its group of origin. Strains from sourdoughs (CCDM 3018, K19-3) and raw cheese (L12, L24, L32) strongly inhibited the highest number of yeast strains on medium with reconstituted milk. These strains showed a consistent spectrum of genes belonging to class 2a bacteriocins, the gene of chitinase and its extracellular product 9 LACO Chitin-binding protein. Strain CCDM 3018 with the spectrum of class 2a bacteriocin gene, chitinase and significant production of lactic acid in all media performed significant antifungal effects in artificial and reconstituted milk-based media.

In vitro assessment of histamine and lactate production by a multi-strain synbiotic

Recent studies suggest histamine and d-lactate may negatively impact host health. As excess histamine is deleterious to the host, the identification of bacterial producers has contributed to concerns over the consumption of probiotics or live microorganisms in fermented food items. Some probiotic products have been suspected of inducing d-lactic-acidosis; an illness associated with neurocognitive symptoms such as ataxia. The goals of the present study were to test the in vitro production of histamine and d-lactate by a 24-strain daily synbiotic and to outline methods that others can use to test for their production. Using enzymatic based assays, no significant production of histamine was observed compared to controls (P > 0.05), while d-lactate production was comparable to a commercially available probiotic with no associated health risk. These assays provide a means to add to the safety profile of synbiotic and probiotic products.

Sustainable and efficient protocols for in vitro germination and antioxidants production from seeds of the endangered species Araucaria araucana

Background The Pehuén or Monkey puzzle tree (Araucaria araucana) is an evergreen coniferous tree, which has been historically used for social, medicinal, and nutritional purposes. We have recently showed the value of A. araucana seeds as a rich source of micronutrients and antioxidants. This endemic species present in a reduced area in Argentina and Chile is endangered because of the low germination rate and the overexploitation of its edible seeds. Thus, the massive extraction of its seeds is ecologically non-viable resulting in limited availability of its active metabolites. However, biotechnological approaches are attractive strategies of production of valuable metabolites and healthy specimens of endangered plants. The aim of this work was to develop a protocol for in vitro production of antioxidants derived from A. araucana seeds and to obtain healthy plants by optimized seed germination. Results Calli of Pehuén seeds were induced in Murashige and Skoog medium with different combinations of auxins and cytokinins, in light and dark conditions. Callus from embryonic axes developed in medium with 1 mg/l α-naphthaleneacetic acid and 1.5 mg/l 6-benzylaminopurine in light conditions had efficient biomass production, antioxidant activity, high phenolic, and flavonoid content and no cytotoxicity on mammalian cells. Additionally, 100 % germination was obtained in vitro and healthy plants were acclimatized to non-sterile conditions. Conclusion In conclusion, in vitro culture of A. araucana could provide new and sustainable options for production of its valuable metabolites with possible therapeutic and nutritional uses. Also, optimized plant germination and acclimatization of endangered species can contribute to the preservation of pristine environments.

​Effect of Season on Quantity and Competence of Oocytes Recovered Transvaginally from Holstein Cows for In vitro Fertilization

Background: Season of the year can affect the reproductive behavior in Holstein cows, altering the competition of the oocytes, reflecting a reduced production of embryos. The objective of this study was to evaluate the average of total oocytes, competition of oocytes and embryos in the in vitro production process at different season of the year in Holstein cows. Methods: During the four seasons of the year, was performed on each of the oocyte donor cows (winter, n = 957; spring, n = 1571; summer, n = 1776; autumn, n = 1128), by in vivo transvaginal follicular aspiration technique after the collection were subjected to the embryos production in vitro. Result: The highest number of total embryos were produced in winter and autumn, compared to spring and summer (3.76±0.16 and 3.54±0.18 vs. 2.73±0.11 and 2.45±0.10; respectively, P less than 0.05). During winter, a higher percentage of oocyte competition was observed, followed by autumn and spring and less competition shown in summer (26.03±0.39, 19.08±0.29, respectively, P less than 0.05). The quantity and competence of the oocytes collected and in vitro embryo production were drastically reduced during the hottest months of the year in this area of intense heat.

Challenges and Considerations during In Vitro Production of Porcine Embryos

Genetically modified pigs have become valuable tools for generating advances in animal agriculture and human medicine. Importantly, in vitro production and manipulation of embryos is an essential step in the process of creating porcine models. As the in vitro environment is still suboptimal, it is imperative to examine the porcine embryo culture system from several angles to identify methods for improvement. Understanding metabolic characteristics of porcine embryos and considering comparisons with other mammalian species is useful for optimizing culture media formulations. Furthermore, stressors arising from the environment and maternal or paternal factors must be taken into consideration to produce healthy embryos in vitro. In this review, we progress stepwise through in vitro oocyte maturation, fertilization, and embryo culture in pigs to assess the status of current culture systems and address points where improvements can be made.