Vitrification as an Alternative Approach for Sperm Cryopreservation in Marine Fishes.

The Southern Flounder Paralichthys lethostigma is a high-value species and a promising aquaculture candidate. Because sperm volume can be limited in this species (<500 µL), new sperm cryopreservation methods need to be evaluated. Vitrification is an alternative to conventional slow-rate freezing, whereby small volumes are cryopreserved at high cooling rates (>1,000°C/min). The goal of this work was to develop a standardized approach for vitrification of Southern Flounder sperm. The specific objectives were to (1) evaluate thawing methods and vitrification solutions, (2) evaluate the postthaw membrane integrity of sperm vitrified in different cryoprotectant solutions, (3) examine the relationship between membrane integrity and motility, and (4) evaluate the ability of vitrified sperm to fertilize eggs. From the vitrification solutions tested, the highest postthaw motility (28 ± 9% [mean ± SD]) and membrane integrity (11 ± 4%) was observed for 20% ethylene glycol plus 20% glycerol. There was no significant difference in postthaw motility of sperm thawed at 21°C or at 37°C. Fertilization from vitrified sperm in one trial yielded the same fertilization rate (50 ± 20%) as the fresh sperm control, while the sperm from the other two males yielded 3%. This is the first report of fertilization by vitrified sperm in a marine fish. Vitrification can be simple, fast, inexpensive, performed in the field, and, at least for small fishes, offers an alternative to conventional cryopreservation. Because of the minute volumes needed for ultrarapid cooling, vitrification is not presently suited as a production method for large fishes. Vitrification can be used to reconstitute lines from valuable culture species and biomedical models, conserve mutants for development of novel lines for ornamental aquaculture, and transport frozen sperm from the field to the repository to expand genetic resources.

Electrochemical Sensor for Hydrogen Peroxide Based on Prussian Blue Electrochemically Deposited at the TiO2-ZrO2-Doped Carbon Nanotube Glassy Carbon-Modified Electrode.

In this investigation, a hydrogen peroxide (H2O2) electrochemical sensor was evaluated. Prussian blue (PB) was electrodeposited at a glassy carbon (GC) electrode modified with titanium dioxide- and zirconia-doped functionalized carbon nanotubes (TiO2.ZrO2-fCNTs), obtaining the PB/TiO2.ZrO2-fCNTs/GC-modified electrode. The morphology and structure of the nanostructured material TiO2.ZrO2-fCNTs was characterized by transmission electron microscopy, the specific surface area was determined via Brunauer-Emmett-Teller, X-ray diffraction, thermogravimetric analysis, and Fourier transform infrared spectroscopy. The electrochemical properties were studied by cyclic voltammetry and chronoamperometry. Titania-zirconia nanoparticles (5.0 ± 2.0 nm) with an amorphous structure were directly synthesized on the fCNT walls, aged during periods of 20 days, obtaining a well-dispersed distribution with a high surface area. The results indicated that the TiO2.ZrO2-fCNT-nanostructured material exhibits good electrochemical properties and could be tunable by enhancing the modification conditions and method of synthesis. Covering of the nanotubes with TiO2-ZrO2 nanoparticles is one of the main factors that affected immobilization and sensitivity of the electrochemical biosensor. The electrode modified with TiO2-ZrO2 nanoparticles with the 20-day aging time was superior regarding its reversibility, electric communication, and high sensitivity and improves the immobilization of the PB at the electrode. The fabricated sensor was used in the detection of H2O2 in whey milk samples, presenting a linear relationship from 100 to 1,000 µmol L-1 between H2O2 concentration and the peak current, with a quantification limit (LQ) of 59.78 µmol L-1 and a detection limit (LD) of 17.93 µmol L-1.

Production of channel catfish with sperm cryopreserved by rapid non-equilibrium cooling.

This report describes the feasibility of using vitrification for fish sperm. Vitrification can be used to preserve samples in the field and offers an alternative to conventional cryopreservation, although it has not been systematically studied for sperm of aquatic species. The overall goal of the project was to develop streamlined protocols that could be integrated into a standardized approach for vitrification of aquatic species germplasm. The objectives of the present study in channel catfish (Ictalurus punctatus) were to: (1) evaluate the acute toxicity of 5%, 10%, 20% and 30% methanol, N,N-dimethyl acetamide, dimethyl sulfoxide, 1,2-propanediol, and methyl glycol; (2) evaluate a range of devices commonly used for cryopreservation and vitrification of mammalian sperm; (3) compare vitrification with and without cryoprotectants; (4) evaluate the post-thaw membrane integrity of sperm vitrified in different cryoprotectant solutions, and (5) evaluate the ability of vitrified sperm to fertilize eggs. Cryoprotectant concentrations of higher than 20% were found to be toxic to sperm. Methanol and methyl glycol were the least toxic at a concentration of 20% with an exposure time of less than 5 min. We evaluated a method reported for human sperm, using small volumes in loops (15 µl) or cut standard straws (20 µl) with and without cryoprotectants plunged into liquid nitrogen. Cryoprotectant-free vitrification using loops did not yield fertilization (assessed by neurulation), and the fertilization rates observed in two trials using the cut standard straws were low (~2%). In general, fertilization values for vitrification experiments were low and the use of low concentrations of cryoprotectants yielded lower fertilization (<10%) than the use of vitrification solutions containing high cryoprotectant concentrations (as high as 25%). The highest neurulation obtained was from a mixture of three cryoprotectants (20% methanol+10% methyl glycol+10% propanediol) with a single-step addition. This was reflected in the flow cytometry data from which the highest membrane integrity using loops was for 20% methanol+10% methyl glycol+10% propanediol (~50%). We report the first successful sperm vitrification in fish and production of offspring from vitrified sperm in channel catfish. Although the fertilization values were low, at present this technique could nevertheless be used to reconstitute lines (especially in small aquarium fishes), but it would require improvement and scaling up before being useful as a production method for large-bodied fishes such as catfish.

Colloid cysts of the third ventricle in children.

OBJECTIVE: The rarity of colloid cysts in children makes it difficult to characterize this entity and offer meaningful advice on treatment. Infrequent case reports exist, but to date there has been no age-specific assessment. The purpose of this study was to define any differences between children and adults who are evaluated and treated for colloid cysts of the third ventricle. METHODS: Patients with colloid cysts were reviewed and stratified by age. Individuals ≤ 18 years of age were defined as pediatric patients and those > 18 years of age as adults. Clinical and radiographic data, treatment, and postoperative outcomes were compared between both groups. Bivariate analysis was conducted using the Fisher exact test for categorical variables and Mann-Whitney U-test for continuous variables. RESULTS: Of 132 endoscopic resections (121 primary, 10 secondary, and 1 tertiary) of a colloid cyst, 9 (6.8%) were performed in pediatric patients (mean age 14.1 years, range 9-18 years) and 123 (93.2%) were performed in adult patients (mean age 43.8 years, range 19-73 years). Cases were found incidentally more commonly in pediatric than adult patients (66.7% vs 37.4%, p > 0.05), and pediatric patients had lower rates of hydrocephalus than adult patients (11.1% vs 63.4%, p < 0.05). Acute decompensation at presentation was found in 8 adults (6.5%) but no children. Complete cyst removal (88.9% vs 90.2%, p > 0.05) and length of stay (1.6 days vs 2.9 days, p > 0.05) were not significantly different between the groups. Postoperative complications (6.5% in adults, 0% in children) and recurrence (2.4% in adults, 0% in children) were rare in both groups, and there were no treatment-related deaths. The mean postoperative radiological follow-up was longer in pediatric patients (45 months, range 4-89 months) than adults (44.1 months, range 1-171 months). CONCLUSIONS: While differences exist between children and adults regarding colloid cyst presentation, these are in keeping with the predicted evolution of a slow-growing lesion. Consistent with this observation, children had lower rates of hydrocephalus and a smaller mean maximal cyst diameter. Contrary to the published literature, however, sudden deterioration was not observed in pediatric patients but occurred in adult patients. In this limited pediatric sample size, the authors have not recorded any postoperative complications or recurrences to date. These encouraging results with endoscopic removal may positively impact future decisions related to children given their protracted life expectancy and projected rates of progression.

Peroxide Electrochemical Sensor and Biosensor Based on Nanocomposite of TiO2 Nanoparticle/Multi-Walled Carbon Nanotube Modified Glassy Carbon Electrode.

A hydrogen peroxide (H2O2) sensor and biosensor based on modified multi-walled carbon nanotubes (CNTs) with titanium dioxide (TiO2) nanostructures was designed and evaluated. The construction of the sensor was performed using a glassy carbon (GC) modified electrode with a TiO2-CNT film and Prussian blue (PB) as an electrocalatyzer. The same sensor was also employed as the basis for H2O2 biosensor construction through further modification with horseradish peroxidase (HRP) immobilized at the TiO2-fCNT film. Functionalized CNTs (fCNTs) and modified TiO2-fCNTs were characterized by Transmission Electron Microscopy (TEM), Fourier Transform Infrared Spectroscopy (FTIR), and X-Ray DifFraction (XRD), confirming the presence of anatase over the fCNTs. Depending on the surface charge, a solvent which optimizes the CNT dispersion was selected: dimethyl formamide (DMF) for fCNTs and sodium dodecylsulfate (SDS) for TiO2-fCNTs. Calculated values for the electron transfer rate constant (ks) were 0.027 s-1 at the PB-fCNT/GC modified electrode and 4.7 × 10-4 s-1 at the PB-TiO2/fCNT/GC electrode, suggesting that, at the PB-TiO2/fCNT/GC modified electrode, the electronic transfer was improved. According to these results, the PB-fCNT/GC electrode exhibited better Detection Limit (LD) and Quantification Limit (LQ) than the PB-TiO2/fCNT/GC electrode for H2O2. However, the PB film was very unstable at the potentials used. Therefore, the PB-TiO2/fCNT/GC modified electrode was considered the best for H2O2 detection in terms of operability. Cyclic Voltammetry (CV) behaviors of the HRP-TiO2/fCNT/GC modified electrodes before and after the chronoamperometric test for H2O2, suggest the high stability of the enzymatic electrode. In comparison with other HRP/fCNT-based electrochemical biosensors previously described in the literature, the HRP-fCNTs/GC modified electrode did not show an electroanalytical response toward H2O2.

Progress and challenges of fish sperm vitrification: A mini review.

To survive low temperature is required for a long-term storage (cryopreservation), cells should be vitrified to a state in which intracellular water is solidified without ice crystal formation. Two different approaches are described for fish sperm cryopreservation: 1) sperm conventional cryopreservation, in which extracellular water is partially crystallized and 2) sperm vitrification, in which both intra- and extra-cellular liquids are vitrified. Sperm vitrification has been applied to some fish species with limited success. Traditional vitrification requires rapid cooling/warming rates, small sample carriers, and using high permeable cryoprotectant concentrations. The latter cause cytotoxic effects which must be well managed and will require continuous effort to match an appropriate cryoprotectant with suitable apparatus and warming methods. Novel cryoprotectant-free sperm vitrification approach has been applied to several fishes. This review summarizes development of basic procedures and discusses advantages and disadvantages of vitrification when applied it to fish sperm.

Vitrification of Sperm from Marine Fishes: Effect on Motility and Membrane Integrity.

Our goal was to develop a standardized approach for sperm vitrification of marine fishes that can be applied generally in aquatic species. The objectives were to: 1) estimate acute toxicity of cryoprotectants over a range of concentrations; 2) evaluate the properties of vitrification solutions (VS); 3) evaluate different thawing solutions, and 4) evaluate sperm quality after thawing by examination of motility and membrane integrity. Sperm were collected from red snapper (Lutjanus campechanus), spotted seatrout (Cynoscion nebulosus), and red drum (Sciaenops ocellatus). A total of 29 combinations of cryoprotectants were evaluated for toxicity and glass formation. Samples were loaded onto 10-µL polystyrene loops and plunged into liquid nitrogen. There was a significant difference (P < 0.05) in post-thaw motility among VS and among species when using the same VS. The sperm in VS of 15% DMSO + 15% ethylene glycol + 10% glycerol + 1% X-1000™ + 1% Z-1000™ had an average post-thaw motility of 58% and membrane integrity of 19% for spotted seatrout, 38% and 9% for red snapper, and 30% and 19% for red drum. Adaptations by marine fish to high osmotic pressures could explain the survival in the high cryoprotectant concentrations. Vitrification offers an alternative to conventional cryopreservation.

Sperm cryopreservation in live-bearing Xiphophorus fishes: offspring production from Xiphophorus variatus and strategies for establishment of sperm repositories.

Cryopreservation of sperm from Xiphophorus fishes has produced live young in three species: X. hellerii, X. couchianus, and X. maculatus. In this study, the goal was to establish protocols for sperm cryopreservation and artificial insemination to produce live young in X. variatus, and to identify needs for repository development. The objectives were to: 1) collect basic biological characteristics of males; 2) cryopreserve sperm from X. variatus, 3) harvest live young from cryopreserved sperm, and 4) discuss the requirements for establishment of sperm repositories. The 35 males used in this study had a body weight of 0.298±0.096 g (mean±SD), body length of 2.5±0.2 cm, and testis weight of 6.4±3.4 mg. The sperm production per gram of testis was 2.33±1.32×10(9) cells. After freezing, the post-thaw motility decreased significantly to 37%±17% (ranging from 5% to 70%) (p=0.000) from 57%±14% (40%-80%) of fresh sperm (N=20). Artificial insemination of post-thaw sperm produced confirmed offspring from females of X. hellerii and X. variatus. This research, taken together with previous studies, provides a foundation for development of strategies for sperm repositories of Xiphophorus fishes. This includes: 1) the need for breeding strategies for regeneration of target populations, 2) identification of minimum fertilization capacity of frozen samples, 3) identification of fish numbers necessary for sampling and their genetic relationships, 4) selection of packaging containers for labeling and biosecurity, 5) assurance of quality control and standardization of procedures, 6) information systems that can manage the data associated with cryopreserved samples, including the genetic data, 7) biological data of sampled fish, 8) inventory data associated with frozen samples, and 9) data linking germplasm samples with other related materials such as body tissues or cells saved for DNA and RNA analyses.

Preserving and using germplasm and dissociated embryonic cells for conserving Caribbean and Pacific coral.

Coral reefs are experiencing unprecedented degradation due to human activities, and protecting specific reef habitats may not stop this decline, because the most serious threats are global (i.e., climate change), not local. However, ex situ preservation practices can provide safeguards for coral reef conservation. Specifically, modern advances in cryobiology and genome banking could secure existing species and genetic diversity until genotypes can be introduced into rehabilitated habitats. We assessed the feasibility of recovering viable sperm and embryonic cells post-thaw from two coral species, Acropora palmata and Fungia scutaria that have diffferent evolutionary histories, ecological niches and reproductive strategies. In vitro fertilization (IVF) of conspecific eggs using fresh (control) spermatozoa revealed high levels of fertilization (>90% in A. palmata; >84% in F. scutaria; P>0.05) that were unaffected by tested sperm concentrations. A solution of 10% dimethyl sulfoxide (DMSO) at cooling rates of 20 to 30°C/min most successfully cryopreserved both A. palmata and F. scutaria spermatozoa and allowed producing developing larvae in vitro. IVF success under these conditions was 65% in A. palmata and 53% in F. scutaria on particular nights; however, on subsequent nights, the same process resulted in little or no IVF success. Thus, the window for optimal freezing of high quality spermatozoa was short (∼5 h for one night each spawning cycle). Additionally, cryopreserved F. scutaria embryonic cells had∼50% post-thaw viability as measured by intact membranes. Thus, despite some differences between species, coral spermatozoa and embryonic cells are viable after low temperature (-196°C) storage, preservation and thawing. Based on these results, we have begun systematically banking coral spermatozoa and embryonic cells on a large-scale as a support approach for preserving existing bio- and genetic diversity found in reef systems.

Production of F1 offspring with vitrified sperm from a live-bearing fish, the green swordtail Xiphophorus hellerii.

This study reports the first production of offspring with vitrified sperm from a live-bearing fish Xiphophorus hellerii. The overall goal of this study was to develop streamlined protocols for integration into a standardized approach for vitrification of aquatic species germplasm. The objectives were to (1) estimate acute toxicity of cryoprotectants, (2) evaluate vitrification solutions, (3) compare different thawing methods, (4) evaluate membrane integrity of post-thaw sperm vitrified in different cryoprotectants, and (5) evaluate the fertility of vitrified sperm. Nine cryoprotectants and two commercial vitrification additives were tested for acute toxicity and glass forming ability, alone and in combination. Two vitrification solutions, 40% glycerol (Gly) and 20% Gly+20% ethylene glycol (EG) in 500 mOsmol/kg Hanks' balanced salt solution (HBSS), were selected for vitrification of 10 µL sperm samples using inoculating loops plunged into liquid nitrogen. Samples were thawed at 24°C (one loop in 5 µL of HBSS or three loops in 500 µL of HBSS). Samples thawed in 500 µL were concentrated by centrifugation (1000 g for 5 min at 4°C) into 5 µL for artificial insemination. Offspring were produced from virgin females inseminated with sperm vitrified with 20% Gly+20% EG and concentrated by centrifugation.

Antrax en dos comunidades de Zacatecas, México / Anthrax in 2 communities of Zacatecas, Mexico

En el estado de Zacatecas se dispone de pocos datos epidemiológicos y epizootiológicos del ántrax y se desconoce la magnitud real de la enfermedad. Entre 1981 y 1983 se presentaron tres pequeños brotes epidémicos que afectaron a 34 personas en dos municipios. Todos los casos de ántrax en humanos se relacionaron con defunciones recientes de bovinos infectados que se habían destazado para ingerir su carne. La enfermedad fue más frecuente entre los hombres (55,8%) que entre las mujeres (44,1%) y el grupo de edad más afectado fue el de 35 a 54 años. La sintomatología fue semejante en todos los casos y se caracterizó por lesiones con pústula negra. La mayoría de los enfermos habían recibido tratamiento médico o se medicaban con remedios caseros cuando se detectaron. Algunos pacientes manifestaron dolor, malestar y fiebre, lo que puede atribuirse a complicaciones causadas por un tratamiento inadecuado ya que en algunos casos se prescribieron antiinflamatorios, pomadas exfoliantes e, incluso, compresas calientes. Aproximadamente la mitad de los enfermos se habían quemado las heridas por diversos procedimientos que son de uso común en la región como medida terapéutica. Los Servicios Coordinados de Salud Pública en el estado de Zacatecas proporcionaron tratamiento médico con penicilina procaína a razón de 800 000 U cada 12 horas hasta que desaparecieron las lesiones. No hubo ningún caso mortal en humanos. Por su parte, la Dirección de Ganadería de la Secretaría de Agricultura y Recursos Hidráulicos emprendió la vacunación de los animales susceptibles de contraer la enfermedad y orientó a las comunidades sobre la incineración y el enterramiento de los animales muertos. Sin embargo, las disposiciones sobre el control de los animales resultan poco eficaces debido a que las coberturas de vacunación son bajas y no se aplican medidas adecuadas con respecto a los pastizales contaminados donde pasta el ganado