Biotechnological Procedures Research Articles

Factors influencing ovum pick-up technique results in cattle

The Ovum Pick-Up (OPU) technique, which is used in in vitro embryo production (IVP) to retrieve immature oocytes from live donor animals, is one of the most important biotechnological procedures used in cattle breeding. The most important advantage of this technology is that it allows for the reproducible retrieval of immature oocytes from living donor animals. It is particularly useful in dairy cattle breeding to address infertility issues and boost the production of superior animals with high genetic value. The OPU technique offers several advantages, including its applicability to cows ranging from six-month-old calves to the first three months of pregnancy, its effectiveness in animals with genital tract infection or acyclic cattle, and its ability to yield a higher number of embryos within the same period compared to the Multiple Ovulation and Embryo Transfer (MOET) technique. Understanding and improving the technical and biological factors influencing the OPU procedure is necessary to increase and optimize donor animal use in IVP and the number of quality oocytes obtained. This review aims to examine the specifics of the OPU approach and the factors influencing its performance in light of contemporary literature, as well as to propose fresh ideas to researchers.

Promoting Environmental Sustainability: Innovative Project Funded by AP Vojvodina

The Autonomous Province (AP) of Vojvodina in Serbia is blessed with abundant surface water resources yet suffers from significant organic pollution due to inadequate waste management practices. In line with AP Vojvodina’s sustainability strategy, this project focuses on developing innovative technologies for managing industrial waste and removing organic pollutants from water resources. The main objective is to create an efficient and environmentally sustainable solution for integrated industrial effluent management and organic pollutant removal in AP Vojvodina. Specific goals include the development of biotechnological procedures for utilizing industrial effluents in the production of xanthan, biopolymer with potential photocatalytic activity for decomposing organic substances in natural waters. By promoting sustainable materials and technologies, the project aims to advance scientific and technological knowledge in biopolymer production and water purification, fostering regional scientific and industrial growth. The project's outcomes are expected to enhance researchers' visibility, facilitate international cooperation, and contribute to the ecological, economic, and social sustainability of AP Vojvodina.

Impact of biotechnological transgenesis procedures on duck productivity

The use of poultry as a unique model of biological research was characterised by a high level of efficiency, however, methods for creating transgenic ducks, complicated by the structure of waterfowl eggshells, are of low efficiency. The purpose of the study was to determine the influence of various biotechnological procedures for creating transgenic ducks on their productive qualities and reproductive ability to identify the optimal method for creating transgenic poultry for further use in scientific, research, or economic purposes. Weighting, morphometric and statistical analysis of productive traits were used during the study. 40 ducks (4 experimental groups of animals and about 3,000 of their eggs) were studied. The lowest value of the egg productivity index was obtained in the group created by busulfan injection (79.5±11.8%), the highest – in the group created by sperm-mediated gene transfer (91.8±2.3%), the group of direct injection of transgenic construct – 89.0±2.0%, which indicates that this biotechnological method of introducing transgenic construct did not have a clear effect on this indicator. The weight of ducks in different experimental groups ranged from 1,323.50±65.36 g (using the sperm-mediated gene transfer) to 1,608.08±94.76 g (in the group created using busulfan). Ducks that received direct injections had an average weight of 1,480.42±35.01 g. In the control group, the average weight at sexual maturity was 139.5±9.67 g, in the busulfan group – 148.2±13.13 g, in the direct injection group – 143.16±7.25 g, and in the spermmediated gene transfer group – 140.67±13.13 g. It was found that the method of injection into the embryo of a recipient sterilised with busulfan and the introduction of donor blastodermal cells negatively affect the reproductive qualities of ducks. The practical significance of the study lies in the fact that as a result of the analysis of the productivity of ducks obtained by various methods of transgenesis, it was determined that the most effective of the evaluated methods is the transfection of DNA of the transgenic construct with sperm (Sperm-mediated gene transfer, SMGT)

Gas Chromatography-Mass Spectrometry Profiling of Volatile Metabolites Produced by Some Bacillus spp. and Evaluation of Their Antibacterial and Antibiotic Activities.

Bacillus species produce different classes of antimicrobial and antioxidant substances: peptides or proteins with different structural compositions and molecular masses and a broad range of volatile organic compounds (VOCs), some of which may serve as biomarkers for microorganism identification. The aim of this study is the identification of biologically active compounds synthesized by five Bacillus species using gas chromatography coupled to mass spectrometry (GC-MS). The current study profoundly enhances the knowledge of antibacterial and antioxidant metabolites ensuring the unambiguous identification of VOCs produced by some Bacillus species, which were isolated from vegetable samples of potato, carrot, and tomato. Phylogenetic and biochemical studies were used to identify the bacterial isolates after culturing. Phylogenetic analysis proved that five bacterial isolates BSS12, BSS13, BSS16, BSS21, and BSS25 showed 99% nucleotide sequence similarities with Bacillus safensis AS-08, Bacillus cereus WAB2133, Bacillus acidiproducens NiuFun, Bacillus toyonesis FORT 102, and Bacillus thuringiensis F3, respectively. The crude extract was prepared from bacterial isolates to assess the antibiotic resistance potency and the antimicrobial potential against various targeted multidrug-resistant strains, including yeast strains such as Candida albicans, Candida krusei, and bacterial strains of Enterococcus hirae, Escherichia coli, Klebsiella aerogenes, Klebsiella pneumoniae, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus group B, Streptococcus mutans, Shigella sonnei, Salmonella enteritidis, Serratia marcescens, Pseudomonas aeruginosa, and Proteus vulgaris. GC-MS analysis of bacterial strains found that VOCs from Bacillus species come in a variety of chemical forms, such as ketones, alcohols, terpenoids, alkenes, etc. Overall, 69 volatile organic compounds were identified from five Bacillus species, and all five were found to share different chemical classes of volatile organic components, which have a variety of pharmacological applications. However, eight antibacterial compounds with different concentrations were commonly found in all five species: acetoin, acetic acid, butanoic acid, 2-methyl-, oxime-, methoxy-phenyl, phenol, 1,2-benzenedicarboxylic acid, bis(2-methylpropyl) ester, nonanoic acid, and hexadecanoic acid, methyl. The present study has demonstrated that bacterial isolates BSS25, BSS21, and BSS16 display potent inhibitory effects against Candida albicans, while BSS25, BSS21, and BSS13 exhibit the ability to restrain the growth and activity of Candida krusei. Notably, BSS25 and BSS21 are the only isolates that demonstrate substantial inhibitory activity against Klebsiella aerogenes. This disparity in inhibitory effects could be attributed to the higher concentrations of acetoin in BSS25 and BSS21, whereas BSS16 and BSS13 have relatively elevated levels of butanoic acid, 2-methyl-. Certainly, the presence of acetoin and butanoic acid, 2-methyl-, contributes to the enhanced antibacterial potential of these bacterial strains, in conjunction with other organic volatile compounds and peptides, among other factors. The biology and physiology of Bacillus can be better understood using these results, which can also be used to create novel biotechnological procedures and applications. Moreover, because of its exceptional ability to synthesize and produce a variety of different antibacterial compounds, Bacillus species can serve as natural and universal carriers for antibiotic compounds in the form of probiotic cultures and strains to fight different pathogens, including mycobacteria.

The investigation of post-thaw chilled storage and the applicability of large-scale cryopreservation in chub (Squalius cephalus) sperm

Chub (reophillic cyprinids) is one of the most sensitive bioindicator fish of environmental changes following anthropogenic activities. The improvement of different biotechnological procedures could help support its conservation and strengthen the natural populations. The aim of this study was to compare the effects of two different hormonal agents (carp pituitary extract and Ovopel™) on various motility parameters (pMOT-%, DAP-μm, VCL μm s−1, VSL-μm s−1, LIN-%, ALH-μm, BCF-Hz) of fresh and cryopreserved/thawed sperm (stored at 4 °C for 6 h). Additionally, we sought to develop a novel, large-scale cryopreservation method for chub sperm, assessing freezing methods (Styrofoam box and a controlled-rate freezer) and different containers (0.5, 5 mL straw and 4 mL cryotube) for sperm cryopreservation. The results of this study indicated no difference between the carp pituitary extract and Ovopel treated groups in either the fresh or frozen/thawed sperm (at 0, 3, 6, hour post thawing, P = 0.4351). In contrast, the quality of the thawed chub sperm was negatively affected after 3 h chilled storage in both hormonal treatments (P = 0.0036, P < 0.0001). When assessing the motility parameters of the sperm between the 5 mL straw and 4 mL cryotube groups cryopreserved in a Styrofoam Box, no difference was observed (P = 0.103). Additionally, sperm loaded in 4 mL cryotubes showed no difference in motility when cryopreserved with either the Styrofoam box or controlled-rate freezer methods (P = 0.109). A similar hatching rate was observed in sperm preserved using the Styrofoam box (35 ± 7 %) and controlled rate freezer (25 ± 9 %) methods (P = 0.300). In a second fertilization trial, hatching rate was similar between control (72 ± 19 %) and cryopreserved (4 mL cryotube and Styrofoam box, 61 ± 5 %) groups. (P = 0.257). Based on our findings and its standard features (less species specific, precise dose calculation), Ovopel can be a good candidate for the stimulation of spermiation in chub sperm prior to cryopreservation. Furthermore, our study presents a novel and applicable method for the large-scale cryopreservation of chub sperm.

A Promising Approach for Addressing Micronutrient Deficiencies and Enhancing Nutritional Quality in Food Crops through Biofortification

Biofortification is a promising agricultural approach for addressing micronutrient shortages and increasing the nutritional content of food crops. Micronutrient deficiencies, also known as "hidden hunger," continue to affect many people around the world, particularly in underdeveloped countries. This presentation emphasises the need of biofortification in solving this worldwide health issue. Biofortification tries to improve the level of important micronutrients such as iron, zinc, vitamin A, and iodine in edible plant portions by using traditional breeding techniques or current biotechnological procedures. Iron-fortified beans, zinc-enhanced rice, vitamin A-rich sweet potatoes, and iodine-enriched crops are all examples of successful biofortified crops. To maximise their impact, biofortification programmes must be integrated with nutrition education and agronomic practises, according to the abstract. Governments, research institutes, non-governmental organisations, and the commercial sector must work together to scale up biofortification activities and build healthier, more resilient communities around the world.

Dual-Purpose Cattle Raised in Tropical Conditions: What Are Their Shortcomings in Sound Productive and Reproductive Function?

Dual-purpose husbandry might well be the most commonly employed cattle management system in tropical regions worldwide. The advantages of producing both meat and milk, although in reduced quantities, gives an edge to the farmer in coping with the volatile economic conditions that prevail in the region. Herein, we discuss the different methods of cattle management under tropical conditions based on the financial and social structure of this system. An account of the sanitary and nutritional conditions available to the farmers and how these factors affect the profitability of the enterprise will also be given. Finally, we will discuss how these systems can take advantage of several biotechnological procedures, and how these tools (such as controlled natural mating, artificial insemination, and embryo transfer) affect reproductive outcomes. The present review will mainly concentrate on production systems located less than 1000 m above sea level, as the problems and shortcomings of cattle raised above this arbitrary landmark are quite different.

Population-Related Variability in Qualitative and Quantitative Secondary Metabolite Profile of Gentianella austriaca (A. & J. Kern.) Holub.

Phytochemical profiling of six natural populations of Gentianella austriaca was performed by HPLC identification and quantification of a number of secondary metabolites, and evaluation of time series of peak areas by chemometric analysis. Phytochemical analysis of G. austriaca revealed the presence of iridoids, flavone-C-glucosides and xanthones. Twelve secondary metabolites were identified in the aerial parts, roots and seeds, including swertiamarin (SWM), gentiopicrin (GP), sweroside (SWZ), isoorientin (ISOOR), swertisin (SWE), demethylbellidifolin-8-O-glucoside (DMB-8-O-glc), bellidifolin-8-O-glucoside (BDF-8-O-glc), mangiferin (MGF), corymbiferin (CBF), corymbiferin-1-O-glucoside (CBF-1-O-glc), bellidifolin (BDF) and campestroside. Multivariate statistical analyses showed relatively low variability among populations according to secondary metabolite content. However, some pharmacologically important compounds were found in higher amounts in a few populations, which could be useful for conservation and future biotechnological procedures.

Eight Up-Coming Biotech Tools to Combat Climate Crisis.

Biotechnology has a high potential to substantially contribute to a low-carbon society. Several green processes are already well established, utilizing the unique capacity of living cells or their instruments. Beyond that, the authors believe that there are new biotechnological procedures in the pipeline which have the momentum to add to this ongoing change in our economy. Eight promising biotechnology tools were selected by the authors as potentially impactful game changers: (i) the Wood-Ljungdahl pathway, (ii) carbonic anhydrase, (iii) cutinase, (iv) methanogens, (v) electro-microbiology, (vi) hydrogenase, (vii) cellulosome and, (viii) nitrogenase. Some of them are fairly new and are explored predominantly in science labs. Others have been around for decades, however, with new scientific groundwork that may rigorously expand their roles. In the current paper, the authors summarize the latest state of research on these eight selected tools and the status of their practical implementation. We bring forward our arguments on why we consider these processes real game changers.

Review: Synthetic Microbial Consortia in Bioremediation and Biodegradation

Since ancient times, we have worked with microbial consortia in a variety of contexts, including wastewater treatment, the production of biogas, additionally to biodegradation and bio cleansing. The great ability of microbial consortiums is, however, a very long way from being completely realized. Last few years have seen a surge in interest in biosynthesis and bioprocessing related to the understanding and use of microbial consortia. It can be difficult to implement complex tasks in a single population. Synthetic consortiums of microorganisms have long utilized in biotechnology procedures like waste management, agricultural farming, and fermentation. Today, microbial consortiums are being engineered for a range of uses by synthetic biologists. The division of collaborative work in consortia is crucial for the breakdown of environmental contaminants that are persistent, cultures need to be resilient to the complicated environment, which often needs several phases. As a result, bioremediation may greatly benefit from the use of synthetic microbial consortiums [1]. The created and improved synthetic microbial community can operate as a culture (seed culture) for ex situ remediation methods including biodegradation in smaller reactors and bio augmentation of in situ bioremediation practices. In order to prevent genetic contamination from environmental microorganisms, the use of designed microbial consortia is currently, to a large degree, restricted in carefully monitored bioprocesses. In this review, an overview of undefined naturally occurring microbial consortia and their application was discussed. We introduced the notion of synthetic microbial consortia, system biology, we discussed Importance of synthetic microbial consortia with relevant examples of how they add value to bio refineries. We did an overview of microbial consortia in biotechnological process, application of microbial consortia in bioremediation and biodegradation was further discussed.

Fluorescent Single-Core and Multi-Core Nanoprobes as Cell Trackers and Magnetic Nanoheaters

Iron oxide magnetic nanoparticles (MNPs) have been widely studied due to their versatility for diagnosis, tracking (magnetic resonance imaging (MRI)) and therapeutic (magnetic hyperthermia and drug delivery) applications. In this work, iron oxide MNPs with different single-core (8–40 nm) and multi-core (140–200 nm) structures were synthesized and functionalized by organic and inorganic coating materials, highlighting their ability as magnetic nanotools to boost cell biotechnological procedures. Single core Fe3O4@PDA, Fe3O4@SiO2-FITC-SiO2 and Fe3O4@SiO2-RITC-SiO2 MNPs were functionalized with fluorescent components with emission at different wavelengths, 424 nm (polydopamine), 515 (fluorescein) and 583 nm (rhodamine), and their ability as transfection and imaging agents was explored with HeLa cells. Moreover, different multi-core iron oxide MNPs (Fe3O4@CS, Fe3O4@SiO2 and Fe3O4@Citrate) coated with organic (citrate and chitosan, CS) and inorganic (silica, SiO2) shells were tested as efficient nanoheaters for magnetic hyperthermia applications for mild thermal heating procedures as an alternative to simple structures based on single-core MNPs. This work highlights the multiple abilities offered by the synergy of the use of external magnetic fields applied on MNPs and their application in different biomedical approaches.

CHROMOSOMIC STUDIES IN ZEPHYRANTHES CITRINA BAKER (AMARYLLIDACEAE), A POLYPLOID ORNAMENTAL

Zephyranthes citrina is an ornamental American bulbous plant used as an ornamental garden crop for the aesthetic qualities of its yellow perigonium. The objective of this work was to characterize the species by classical chromosome staining and fluorochrome banding. A sporophytic chromosome number of 2n=8x=48 chromosomes was observed, being the karyotypic formula 20 m + 26 sm + 2 st. Satellites were detected in the short arm of metacentric chromosomes 8, 9, 11 and 12, which colocalized with constitutive heterochromatin CMA+/DAPI-/0 bands. The karyotype comprised chromosome pairs with terminal constitutive heterochromatin bands that included satellites and heteromorphic clusters indicating that it is an allooctoploid. These results will be used as a tool for monitoring genetic improvement, in interspecific crosses and its progenies and in biotechnological procedures by in vitro culture. Key words: constitutive heterochromatin, chromosome banding, bulbous, plant genetic resources, karyotype

Relative transcript level of BMP15, BAX and CASP3 with qRT-PCR in vitrified equine immature cumulus-oocytes complexes

Research focused on female gamete vitrification has increased attention to develop a reliable cryopreservation method to preserve immature equine oocytes. Despite the intensive implementation of biotechnological procedures for horse breeding, vitrification of immature equine cumulus-oocyte complexes (COCs) remain to be clearly elucidated. We aimed to determine the relative transcript level of target genes Bone morphogenetic protein 15 (BMP15); Bcl-2-associated X protein (BAX); and Caspase 3 (CASP3) in equine COCs prior to and after vitrification. Ovarian follicles were aspirated from ovaries collected from an abattoir. A total of 240 COCs were collected and distributed into vitrified COCs (VIT, n=120) and non-vitrified (Non-VIT, n=120) groups. Then, COCs were preserved and relative transcript expressions of BMP15, BAX, CASP3 were measured and normalized against GAPDH performed by qRT-PCR. In addition, 38 COCs were evaluated to assess chromatin configuration of germinal vesicl e stage prior and after vitrification by exposure to 10 μg/ml of bisbenzimide. A difference was observed in the COCs’ mRNA level of abundance for the BAX gene between the VIT (2.05 ± 0.47) and (0.85 ± 0.08) Non-VIT groups. There was no difference in mRNA relative transcript level of CASP3 and BMP15 in Non-VIT (0.63 ± 0.20 and 1.55 ± 0.73, respectively) compared to VIT (0.64 ± 0.01 and 2.84 ± 2.20, respectively) equine COCs. All COCs where considered at immature stage of development even though COCs in Non-VIT group showed higher condensed chromatin configuration compared to VIT (100% vs 60.7%, respectively). We demonstrate that BMP15 and CASP3 are detected in VIT and Non-VIT immature COCs. In conclusion, BAX is expressed highly in vitrified immature equine COCs and indicates that activation of apoptosis signaling cascades in cells exposed to vitrification.

Comprehensive strategies of Lignocellulolytic enzyme production from microbes and their applications in various commercial-scale faculties

Activities of anthropological organisms lead to the production of massive lignocellulosic waste every year and these lignocellulolytic enzymes plays crucial role in developing eco-friendly, sustainable and economical methods for decomposing and pre-treating the biomass to produce biofuels, organic acids, feeds and enzymes. Lignocellulolytic enzymes sustainably hydrolyse the biomass and can be utilized in wide range of applications such as personal care, pharmaceutical, biofuel release, sewage treatment, food and beverage industries. Every year a significant ton of biomass waste is released and insight on these crucial enzymes could establish in all the industries. However, due to the increased demand for compost materials, biomass degradation has resulted in composting processes. Several methods for improving compost amount and quality have been explored, including increasing decomposer inoculums, stimulating microbial activity, and establishing a decomposable environment. All of these prerequisites are met by biotechnological applications. Biotechnological procedures are used to improve the activity of enzymes on biomass. It leads to an adequate supply of compost and base materials for enterprises. In terms of effectiveness and stability during the breakdown process, lignocellulolytic enzymes derived from genetically modified species outperformed naturally derived lignocellulolytic enzymes. It has the potential to increase the quality and output of by-products. This review discussed the development of lignocellulolytic enzyme families and their widespread applications in a variety of industries such as olive oil extraction, carotenoid extraction, waste management, pollution control, second-generation bio-ethanol production, textile and dyeing, pharmaceuticals, pulp and paper, animal feed, food processing industries, detergent, and agricultural industries.

Deep learning strategies for active secondary metabolites biosynthesis from fungi: Harnessing artificial manipulation and application

Microorganisms mainly fungi considered the most natural inhabitants of diverse environments exhibit enormous interactions capabilities to produce active molecules. However, these substances are typically produced only in limited amounts by their native hosts. The native organisms may also be difficult to cultivate and genetically engineer, and some can produce undesirable toxic bio-products. The biologically active secondary metabolites from endophytic fungi showed several kinds of bioactivities, comprising antimicrobial, anti-tumor, anti-inflammatory, immunomodulatory and anti-diabetes, developing functional constipation activities. Particularly, the novel biotechnological procedures including co-cultivation and various fermentation strategies to enhance fungal biomass and subsequently increasing valuable active products with maximum yields, have been paid more and more attention by scientists and researchers, but most endophytic fungi appear to be uncultivable, and for those that can be cultivated, their metabolic potential lies largely invisible through regulatory silencing of biosynthetic genes for secondary metabolites production. This review introduced the biologically active metabolites and their main classes from fungi through artificial manipulation methods, effects and substrates, in recent years, and then the relationships between active molecules and their bio activity effects were also discussed.

Approaching transglutaminase from Streptomyces bacteria over three decades.

Transglutaminases are protein cross-linking and protein-modifying enzymes that have attracted considerable interest due to their causal involvement in various diseases and versatility in industrial applications. In particular, microbial transglutaminases (MTG) from Streptomyces bacteria have managed in recent years to evolve from simple food additives to specialized enzymes for the site-directed modification of therapeutic proteins. The review summarizes relevant studies from the beginning dealing with the occurrence, production, structure, catalysis, and substrate molecules of MTG enzymes. It also addresses biotechnological procedures with MTG from S. mobaraensis (SmMTG) as the most prominent representative in focus. Reassessment of the available data revealed unexpected insights into catalysis of SmMTG and other transglutaminases, suggesting selection of glutamine donor proteins by subsites at the front vestibule and the existence of distinct lysine pockets. Flexibility of the SmMTG-accessible glutamine donor substrate regions seems to be more important than the glutamine environment. Nevertheless, residues in close vicinity to glutamines also determine interaction with the SmMTG subsites. The apparent lack of subsites for lysine donor proteins suggests self-assembly of the substrate proteins prior to enzymatic cross-linking. The study of natural substrate proteins, especially their mutual interaction, is proposed to further illuminate catalysis of SmMTG. To this end, structure and function of the characterized substrate proteins from S. mobaraensis are discussed in conclusion.

CURRENT STATUS AND FUTURE PROSPECTS OF ARTIFICIAL INSEMINATION IN GOATS IN NEPAL: A REVIEW

Nepal is endowed with several indigenous, exotic, and crossbred goat species and breeds that carry potency to contribute a far more to agricultural production than they are presently contributing. Low productivity and high demands of goats have been prevailing in Nepal for a long. Detailed knowledge about various biotechnological procedures like artificial insemination (AI) for breeding is vital in managing goat for high reproductive rates and increasing productivity. Problem of inbreeding in goat exists in farms of Nepal. Thus, Goat farmers can use AI for genetic improvement in their herds. This review aims to provide sufficient knowledge about AI in goats, its prevailing condition, and prospects in Nepal. It also aims to disseminate knowledge about reproduction in goats, oestrous synchronization, and many other factors related to AI. Findings revealed that AI is currently being practiced in Nepal to improve flock productivity and carries huge scope for the future too. However, the present status of AI was found unconvincing. The conception rate was just 35% with total 4,499 AI in goats in 2018/19, and the coverage was less than 1%. Studies also showed that the facilities for semen collection, processing, preservation, and storage are available in Nepal. But a huge amount of semen is still being imported because of insufficient technologies here. Limited researcher related to AI in goats in Nepal is also a hindrance to develop AI in goats in Nepal. It is better to conduct a detailed study on AI in goats in Nepal to help farmers, traders, and planners.

Occupational Health and Safety in Biotechnological Processes: A Review and Future Directions

An option to change partially or completely conventional chemical methods is a biotechnological process. It enables the development of environmentally friendly and innovative means. The safety of this process has not been fully examined, thus, the lack of awareness about risk management is a great concern. This work aims to elucidate the importance of recognizing, assessing, and controlling potential risks, and developing appropriate risk management to develop bioprocesses safely. For this purpose, qualitative research was carried out through scientific studies and current legislation. Safe development of biotechnological procedures allows for occupational safety throughout the processes. It was observed that control measures should be adopted to obtain a safe work environment for everyone. These measures can be carried out through anticipation, recognition, assessment, and control of existing risks or that may exist in the process. Also, it was found that the involvement of all adequate risk management is fundamental. Therefore, biotechnological processes must be developed safely for workers, the environment, and society.&#x0D;

The inhibition of spermatic cystine/glutamate antiporter xCT (SLC7A11) influences the ability of cryopreserved stallion sperm to bind to heterologous zonae pellucidae

Sperm are redox-regulated cells, and deregulation of their redox status is considered to affect male fertility and to reduce their fertilizing ability following biotechnological procedures, such as cryopreservation. Cystine (CysS), after incorporation in sperm via SLC7A11 antiporter, has been demonstrated to increase intracellular GSH content, the most important non enzymatic antioxidant. This study was aimed at investigating the role of SLC7A11 antiporter on frozen-thawed stallion sperm ability to respond to in vitro capacitating environment after post-thaw incubation with CysS and/or Sulfasalazine (SS), a specific inhibitor of SLC7A11 antiporter. Viability, motility, immunolocalization of tyrosine phosphorylated proteins and the ability to bind to heterologous zonae pellucidae were evaluated. Thawed sperm from seven stallions (2 ejaculates/stallion) was washed and resuspended in Tyrodes media; each thawed ejaculate was divided in Control (CTR) and 3 samples supplemented with: 0.5 mM Cystine (CysS), 500 μM Sulfasalazine (SS) and 0.5 mM CysS + 500 μM SS (CysS + SS). After 1 h of incubation at 37 °C, samples were washed twice, resuspended in capacitating BWW medium and incubated at 38 °C under 5% CO2. After 30 and 60 min, sperm motility, viability and tyrosine phosphorylated protein immunolocalization, used as capacitation status index, were evaluated. After 30 min of capacitation, 4 × 105 sperm were co-incubated with denuded pig oocytes in capacitation medium for 30 min for the heterologous binding assay. None of the sperm parameters studied (motility, viability and tyrosine phosphorylation) showed any difference respective to control. The number of sperm bound per oocyte (mean ± SEM) tended to increase in CysS group (44.0 ± 12.3) respect CTR (40.8 ± 10.8) while decreased in SS group (32.4 ± 7.8) (p < 0.01). Moreover, CysS + SS group showed a lower binding rate (32.0 ± 10.0) compared to CysS (p < 0.001). Our results suggest that CysS supplementation of thawed stallion sperm can influence their ability to bind to heterologous zona pellucidae as the inhibition of CysS incorporation by SLC7A11 reduced the number of sperm bound per oocyte. This effect does not seem to be ascribed to a modification of sperm motility, membrane integrity and tyrosine phosphorylation.

Applications of gellan natural polymer microspheres in recombinant catechol-O-methyltransferase direct capture from a Komagataella pastoris lysate

The present work shows the application of nickel- and magnesium-crosslinked gellan microspheres in ionic and affinity capture strategies to directly extract hSCOMT from the complex Komagataella pastoris lysate through a simple batch method. Both formulations present similar morphology, but nickel–crosslinked microspheres present higher crosslinker content and smaller diameters. Four different capture strategies were established, by manipulating the ionic strength, pH, temperature and competing agents' presence. The most promising results for hSCOMT capture and clarification were obtained employing an ionic strategy with nickel-crosslinked microspheres and an affinity strategy with magnesium-crosslinked microspheres at 4 °C. The bioactivity results (200%) and purification degree (70%) of hSCOMT captured by the ionic strategy were more satisfactory probably due to the soft ionic conditions used (100 mM NaCl). For the first time, the gellan polysaccharide versatility was demonstrated in the microsphere application for the direct capture of hSCOMT from a complex lysate, simplifying isolation biotechnological procedures.