Effects of different cryopreservation methods on post-thaw culture conditions of in vitro produced bovine embryos

The aim of this work was to evaluate the effect of cryopreservation protocols on subsequent development of in vitro produced bovine embryos under different culture conditions. Expanded in vitro produced blastocysts (n = 600) harvested on days 7-9 were submitted to controlled freezing [slow freezing group: 10% ethylene glycol (EG) for 10 min and 1.2°C/min cryopreservation]; quick-freezing [rapid freezing group: 10% EG for 10 min, 20% EG + 20% glycerol (Gly) for 30 s]; or vitrification [vitrification group: 10% EG for 10 min, 25% EG + 25% Gly for 30 s] protocols. Control group embryos were not exposed to cryoprotectant or cryopreservation protocols and the hatching rate was evaluated on day 12 post-insemination. In order to evaluate development, frozen-thawed embryos were subjected to granulosa cell co-culture in TCM199 or SOFaa for 4 days. Data were analyzed by PROC MIXED model using SAS Systems for Windows?. Values were significant at p < 0.05. The hatching rate of the control group was 46.09%. In embryos cultured in TCM199, slow freezing and vitrification group hatching rates were 44.65 ± 5.94% and 9.43 ± 6.77%, respectively. In embryos cultured in SOFaa, slow freezing and vitrification groups showed hatching rates of 11.65 ± 3.37 and 8.67 ± 4.47%, respectively. In contrast, the rapid freezing group embryos did not hatch, regardless of culture medium. The slow freezing group showed higher hatching rates than other cryopreservation groups. Under such conditions, controlled freezing (1.2°C/min) can be an alternative to cryopreservation of in vitro produced bovine embryos.     

Landscape management and domestication of Stenocereus pruinosus (Cactaceae) in the Tehuacan Valley: human guided selection and gene flow

ABSTRACT: BACKGROUND: Use of plant resources and ecosystems practiced by indigenous peoples of Mesoamerica commonly involves domestication of plant populations and landscapes. Our study analyzed interactions of coexisting wild and managed populations of the pitaya Stenocereus pruinosus, a columnar cactus used for its edible fruit occurring in natural forests, silviculturally managed in milpa agroforestry systems, and agriculturally managed in homegardens of the Tehuacan Valley, Mexico. We aimed at analyzing criteria of artificial selection and their consequences on phenotypic diversity and differentiation, as well as documenting management of propagules at landscape level and their possible contribution to gene flow among populations. METHODS: Semi-structured interviews were conducted to 83 households of the region to document perception of variation, criteria of artificial selection, and patterns of moving propagules among wild and managed populations. Morphological variation of trees from nine wild, silviculturally and agriculturally managed populations was analyzed for 37 characters through univariate and multivariate statistical methods. In addition, indexes of morphological diversity (MD) per population and phenotypic differentiation (PD) among populations were calculated using character states and frequencies. RESULTS: People recognized 15 pitaya varieties based on their pulp color, fruit size, form, flavor, and thorniness. On average, in wild populations we recorded one variety per population, in silviculturally managed populations 1.58 +/- 0.77 varieties per parcel, and in agriculturally managed populations 2.19 +/- 1.12 varieties per homegarden. Farmers select in favor of sweet flavor (71% of households interviewed) and pulp color (46%) mainly red, orange and yellow. Artificial selection is practiced in homegardens and 65% of people interviewed also do it in agroforestry systems. People obtain fruit and branches from different population types and move propagules from one another. Multivariate analyses showed morphological differentiation of wild and agriculturally managed populations, mainly due to differences in reproductive characters; however, the phenotypic differentiation indexes were relatively low among all populations studied. Morphological diversity of S. pruinosus (average MD = 0.600) is higher than in other columnar cacti species previously analyzed. CONCLUSIONS: Artificial selection in favor of high quality fruit promotes morphological variation and divergence because of the continual replacement of plant material propagated and introduction of propagules from other villages and regions. This process is counteracted by high gene flow influenced by natural factors (pollinators and seed dispersers) but also by human management (movement of propagules among populations), all of which determines relatively low phenotypic differentiation among populations. Conservation of genetic resources of S. pruinosus should be based on the traditional forms of germplasm management by local people.     

Safely Discharging Infants with Bronchiolitis from an Emergency Department: A Five Step Guide for Pediatricians

Recent publications have established the pulse oxygen saturation (SpO₂) threshold of 90% for the hospitalization and discharge of infant patients with bronchiolitis. However, there is no clear recommendation regarding the Emergency Department (ED) observation period necessary before allowing safe home discharge for patients with SpO₂ above 90%-92%. Our primary aims were to evaluate the risk factors associated with delayed desaturation in infants with SpO₂ ≥ 92% on arrival at the ED as well as the ED observation period necessary before allowing safe home discharge. A secondary aim was to identify the risk factors for ED readmission. Of 581 episodes of bronchiolitis in patients < 1 year old admitted to the ED, only 47 (8%) had SpO₂ < 92% on arrival there, although 106 (18%) exhibited a delayed desaturation (to < 92%) during ED observation. Female sex, age < 3 months old, ED readmission, more severe initial clinical presentation, and higher pCO₂ level (> 6KPa) were risk factors for delayed desaturation with OR varying from 1.7 to 7.5. In patients < 3 months old, mean desaturation occured later than in older patients [6.0 hours (IQR 3.0–14.0) vs. 3.0 hours (IQR 2.0–6.0), P = 0.0018]. In 95% of patients with a delayed desaturation this decrease occurred within 25 hours for patients < 3 months old and within 11 hours for patients ≥ 3 months old. In patients < 3 months old with respiratory rates above the normal range for their age the desaturation occurred earlier than in patients < 3 months with normal respiratory rates [4.4 hours (IQR 3.0–11.7) vs. 14.6 hours (IQR 7.6–22.2), P = 0.037]. Based on the present study’s results, we propose a five step guide for pediatricians on discharging children with bronchiolitis from the ED. By using the threshold of an 11 hour ED observation period for patients ≥ 3 months old and a 25 hour period for patients < 3 months old we are able to detect 95% of the patients with bronchiolitis who are at risk of delayed desaturation.     

Changes in biooxidation mechanism and transient biofilm characteristics by As(V) during arsenopyrite colonization with <Emphasis Type="Italic">Acidithiobacillus thiooxidans</Emphasis>

Chemical and surface analyses are carried out using Raman spectroscopy, X-ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM–EDS), atomic force microscopy (AFM), confocal laser scanning microscopy (CLSM), glow discharge spectroscopy (GDS) and extracellular surface protein quantification to thoroughly investigate the effect of supplementary As(V) during biooxidation of arsenopyrite by Acidithiobacillus thiooxidans. It is revealed that arsenic can enhance bacterial reactions during bioleaching, which can strongly influence its mobility. Biofilms occur as compact-flattened microcolonies, being progressively covered by a significant amount of secondary compounds (S _n ^2- , S⁰, pyrite-like). Biooxidation mechanism is modified in the presence of supplementary As(V), as indicated by spectroscopic and microscopic studies. GDS confirms significant variations between abiotic control and biooxidized arsenopyrite in terms of surface reactivity and amount of secondary compounds with and without As(V) (i.e. 6 μm depth). CLSM and protein analyses indicate a rapid modification in biofilm from hydrophilic to hydrophobic character (i.e. 1–12 h), in spite of the decrease in extracellular surface proteins in the presence of supplementary As(V) (i.e. stressed biofilms).