Novel Apoplastic Antifreeze Proteins of Deschampsia antarctica as Enhancer of Common Cell Freezing Media for Cryobanking of Genetic Resources, a Preliminary Study.

Antifreeze proteins (AFPs) are natural biomolecules found in cold-adapted organisms that lower the freezing point of water, allowing survival in icy conditions. These proteins have the potential to improve cryopreservation techniques by enhancing the quality of genetic material postthaw. Deschampsia antarctica, a freezing-tolerant plant, possesses AFPs and is a promising candidate for cryopreservation applications. In this study, we investigated the cryoprotective properties of AFPs from D. antarctica extracts on Atlantic salmon spermatozoa. Apoplastic extracts were used to determine ice recrystallization inhibition (IRI), thermal hysteresis (TH) activities and ice crystal morphology. Spermatozoa were cryopreserved using a standard cryoprotectant medium (C+) and three alternative media supplemented with apoplastic extracts. Flow cytometry was employed to measure plasma membrane integrity (PMI) and mitochondrial membrane potential (MMP) postthaw. Results showed that a low concentration of AFPs (0.05 mg/mL) provided significant IRI activity. Apoplastic extracts from D. antarctica demonstrated a cryoprotective effect on salmon spermatozoa, with PMI comparable to the standard medium. Moreover, samples treated with apoplastic extracts exhibited a higher percentage of cells with high MMP. These findings represent the first and preliminary report that suggests that AFPs derived from apoplastic extracts of D. antarctica have the potential to serve as cryoprotectants and could allow the development of novel freezing media.

Preparation and extraction of chorion proteins from Salmo salar embryos at the pigmented eye stage for electrophoresis with SDS-polyacrylamide gel.

The chorion fulfills important functions in fish embryos, including protecting the embryo during development. The characterization of the protein profile of this envelope could be used as a bioindicator in the evaluation of the quality of embryonic development. The object of this work was to validate a standardized protocol for protein extraction from chorion of Salmo salar embryos at 280 accumulated thermal units (ATU) by comparing and combining existing methods. The protocol consists of consecutive washing of the chorion samples followed by protein extraction with the solution that was named SDS solution (Tris-HCl 100 mM (pH 8), Urea 8 M, 1% SDS, ß-mercaptoethanol 300 mM and EGTA 10 Mm, and 1% protease inhibitor cocktail) and mechanical methods. Protein extraction is enhanced by a working temperature of 75 °C and use of a disperser. The protein concentration was quantified by Bradford Assay. After extraction, the samples were diluted (dilution factor 10) before reading against the calibration curve. After gel electrophoresis with a load of 3 µg of protein, staining showed more than 4 bands, with molecular weights between 25 kDa and 180 kDa.•The protein profile of fish chorion was between 25 kDa and 180 kDa.•Solution containing 1% SDS allows a higher extraction of proteins from the chorion of Atlantic salmon embryos with 280 ATU.•Chorion protein identification is a valuable tool in determining gamete and embryo quality in fish.

The Reproductive Biology of Puye (Galaxias maculatus) under Experimental Culture.

This study determines the reproductive patterns of puye (Galaxias maculatus) under culture conditions. A population of 567 wild fish was caught in the Cautín River, Chile, and held in captivity for four years. Mortality, sex ratio, gonadosomatic index (GSI), sexual maturity stages, spawning period, type and frequency of spawning, and fecundity were measured. The fish grew throughout the experimental period, with the fastest rate during the first half of the first year of life. The highest mortality occurred during the first three months of the experiment and during the spawning season. The sex ratio was almost 1:1 (female:male). First sexual maturity was reached at one year of age, with an average weight of 0.85 ± 0.01 g, total length of 4.85 ± 0.16 cm, and condition factor 0.0074. The highest GSI in both females (12.14 ± 0.74) and males (17.7 ± 2.70) was recorded in August. Nevertheless, the females spawned 3 to 10 times between September and February, with the highest reproductive peak between September and October. The number of embryos per female per day varied from 1 to 429, while the total number of embryos per female during the entire season evaluated varied from 163 to 1044. There was a high correlation (r = 0.82) between absolute fecundity and body weight. Although further studies are needed in this field, these results are basic for establishing future reproductive programs in captivity as a strategy for sustainable fisheries and aquaculture management.

Reduced Graphene Oxide as a Platform for the Immobilization of Amino-Cyclodextrins.

In the present work, we reported on a method to combine amino ß-cyclodextrins (CD1) with reduced graphene oxide (obtained by the electrochemical reduction of graphene oxide, erGO) to produce a glassy carbon electrode (GCE) modified with both CD1 and erGO (CD1-erGO/GCE). This procedure avoids the use of organic solvents such as hydrazine or long reaction times and high temperatures. The material combining both CD1 and erGO (CD1-erGO/GCE) was characterized by SEM, ATR-FTIR, Raman, XPS, and electrochemical techniques. As proof-of-concept, the determination of the pesticide carbendazim was carried out. The spectroscopic measurements, especially XPS, proved that CD1 was covalently attached to the surface of the erGO/GCE electrode. The attachment of cyclodextrin at the reduced graphene oxide produced an increase in the electrochemical behavior of the electrode. The cyclodextrin-functionalized reduced graphene oxide, CD1-erGO/GCE, showed a larger sensitivity (1.01 µA/µM) and a lower limit of detection for carbendazim (LOD = 0.50 µM) compared with the non-functionalized material, erGO/GCE, (sensitivity = 0.63 µA/µM and LOD = 4.32 µM, respectively). Overall, the results of the present work show that this simple method is suitable to attach cyclodextrins to graphene oxide, maintaining their inclusion abilities.

Use of Probiotic Bacteria and Bacteriocins as an Alternative to Antibiotics in Aquaculture.

In addition to their use in human medicine, antimicrobials are also used in food animals and aquaculture, and their use can be categorized as therapeutic against bacterial infections. The use of antimicrobials in aquaculture may involve a broad environmental application that affects a wide variety of bacteria, promoting the spread of bacterial resistance genes. Probiotics and bacteriocins, antimicrobial peptides produced by some types of lactic acid bacteria (LAB), have been successfully tested in aquatic animals as alternatives to control bacterial infections. Supplementation might have beneficial impacts on the intestinal microbiota, immune response, development, and/or weight gain, without the issues associated with antibiotic use. Thus, probiotics and bacteriocins represent feasible alternatives to antibiotics. Here, we provide an update with respect to the relevance of aquaculture in the animal protein production sector, as well as the present and future challenges generated by outbreaks and antimicrobial resistance, while highlighting the potential role of probiotics and bacteriocins to address these challenges. In addition, we conducted data analysis using a simple linear regression model to determine whether a linear relationship exists between probiotic dose added to feed and three variables of interest selected, including specific growth rate, feed conversion ratio, and lysozyme activity.

Characterization of Electrospun Poly(ε-caprolactone) Nano/Micro Fibrous Membrane as Scaffolds in Tissue Engineering: Effects of the Type of Collector Used.

Electrospinning is an electrohydrodynamic technique that transforms a polymer solution into nano/microscopic diameter fibers under the influence of a high-voltage electric field. Its use in the fabrication of nano/micro fibrous membranes as scaffolds for tissue engineering has increased rapidly in recent years due to its efficiency and reproducibility. The objective of this study is to show how the use of the same polymeric solution (polycaprolactone 9% w/v in chloroform: isopropanol 50:50) and identical electrohydrodynamic deposition parameters produces fibers with different characteristics using a flat collector platform with movements in the X and Y axes vs. a conventional rotary collector. The manufactured nano/microfibers show significant differences in most of their characteristics (morphology, roughness, hydrophilicity, and mechanical properties). Regarding the diameter and porosity of the fibers, the results were similar. Given that scaffolds must be designed to guarantee adequate survival and the proliferation and migration of a certain cell type, in this study we analyze how the variations in the characteristics of the fibers obtained are essential to defining their potential application.

Bacteriocinogenic probiotic bacteria isolated from an aquatic environment inhibit the growth of food and fish pathogens.

The conditions of aquatic environments have a great influence on the microbiota of several animals, many of which are a potential source of microorganisms of biotechnological interest. In this study, bacterial strains isolated from aquatic environments were bioprospected to determine their probiotic profile and antimicrobial effect against fish and food pathogens. Two isolates, identified via 16S rRNA sequencing as Lactococcus lactis (L1 and L2) and one as Enterococcus faecium 135 (EF), produced a bacteriocin-like antimicrobial substance (BLIS), active against Listeria monocytogenes, Salmonella Choleraesuis and Salmonella Typhimurium. Antimicrobial activity of BLIS was reduced when exposed to high temperatures and proteolytic enzymes (trypsin, pepsin, papain and pancreatin). All strains were sensitive to 7 types of antibiotics (vancomycin, clindamycin, streptomycin, gentamicin, chloramphenicol, rifampicin and ampicillin), exhibited a high rate of adherence to Caco-2 cells and expressed no hemolysin and gelatinase virulence factors. EF showed some resistance at pH 2.5 and 3.0, and L2/EF showed higher resistance to the action of bile salts. Finally, the presence of bacteriocin genes encoding for proteins, including Nisin (L1 and L2), Enterocin A, B, P, and Mundticin KS (EF) was detected. The molecular and physiological evidence suggests that the bacterial isolates in this study could be used as natural antimicrobial agents and may be considered safe for probiotic application.

Characterization of first blastomeres in Patagonian blenny (Eleginops maclovinus) (Perciformes: Eleginopidae).

SummaryThere is no information about the characteristics of early cleavage in the Patagonian blennie (Eleginops maclovinus), which can be used as a diagnostic tool for embryo quality. The purpose of this investigation, therefore, was to characterize the first blastomeres of E. maclovinus morphologically. Of a 'pool' of incubated eggs at 10.7 ± 0.5°C, 100 microphotographs of blastodiscs were extracted at different incubation periods from 0.25 to 5 h after fertilization and analyzed. Blastodiscs taken at 3.5, 4.0 and 5.0 h were characterized and classified into symmetric or asymmetric groups according to their morphology. The proportions of length (L) and width (W) of each blastomere were determined to establish its symmetry. Additionally, 20 microphotographs of blastodiscs of normal appearance were analyzed morphologically (control blastodisc: CB) and compared other blastodiscs (4.0 and 5.0 h). The results showed that before fertilization oocytes presented a somehow turgid aspect (maximum average diameter of 987 ± 41 µm) and after fertilization and hydration, their diameter increased to 1001.5 ± 11 µm (but not statistically significant) and presented a spherical shape. First cleavage ends after 3.5 h of development, forming two blastomeres 467 ± 45 µm length (L) and 328 ± 21 µm width (W) with a L/W ratio of 1.43 ± 0.19. The second cleavage ends after development at 4.5 h forming four blastomeres 238 ± 65 µm length and 227 ± 65 µm width with a ratio L/W of 1.06 ± 0.09. Five categories were identified during the blastomere characterization: 70% normal or symmetric; 8% with odd numbers of blastomeres; 6% unequal; 6% 'pie shaped' and 10% amorphous.

Study of the first blastomeres in Coho salmon (Oncorhynchus kisutch).

Summary There is a lack of information on the morphology of the first blastomeres that could be used as a diagnostic tool for the first stages of embryonic development for Coho salmon. The purpose of this investigation, therefore, was to characterize morphometrically the first blastomeres of Coho salmon (Oncorhynchus kisutch). In total, 660 embryonic discs from a pool of eggs that had been fertilized and incubated at 5°C and after 19 h of incubation were extracted and photographed. Of these, 20 microphotographs of blastodiscs of normal appearance were analyzed morphologically (control blastodiscs: CB) and 100 random microphotographs from the whole group were classified as either symmetrical or asymmetrical according to their morphology and then compared with the CB. The length and width of each blastomere and the proportions of length and width were measured to determine symmetry in the embryos at the 4-cell stage. Seven categories were created to characterize the blastomeres: 38% normal (G1); 26% unequal (G2); 10% 'pie-shaped' (G3); 10% amorphous (G4); 8% with three equal and one unequal blastomere (G5); 6% 'clover-shaped' blastomeres (G7), and 3% with inclusions. The mean of the proportions of lengths and widths of the groups of blastomeres that were measured was 0.87 ± 0.08 and 0.85 ± 0.07, respectively. The morphometric results that were obtained in this investigation are compared with the results observed by other authors for teleostei and are discussed.