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 degrees 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 (R). 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 degrees C/min) can be an alternative to cryopreservation of in vitro produced bovine embryos.

Post-thaw viability of in vivo-produced canine blastocysts cryopreserved by slow freezing

The objectives were to evaluate the reexpansion blastocoele rate, post-thaw viability, and in vitro development of canine blastocysts cryopreserved by slow freezing in 1.0 m glycerol (GLY) or 1.5 m ethylene glycol (EG). Fifty-one in vivo-produced canine blastocysts were randomly allocated in two groups: GLY (n = 26) and EG (n = 25). After thawing, embryos from MO were immediately stained with the fluorescent probes propidium iodide and Hoechst 33 342 to evaluate cellular viability. Frozen-thawed embryos from M3 and M6 were cultured in SOFaa medium + 10% FCS at 38.5 degrees C under an atmosphere of 5% CO2 with maximum humidity, for 3 and 6 days, respectively, and similarly stained. The blastocoele reexpansion rate (24 h after in vitro culture) did not differ between GLY (76.5%) and EG (68.8%). Post-thaw viable cells rate were not significantly different between GLY and EG (66.5 +/- 4.8 and 57.3 +/- 4.8, respectively, mean +/- SEM), or among MO (62.3 +/- 5.7%), M3 (56.9 +/- 6.0%), and M6 (66.5 +/- 6.0%). In conclusion, canine blastocysts cryopreserved by slow freezing in 1.0 m glycerol or 1.5 m ethylene glycol, had satisfactory blastocoele reexpansion rates, similar post-thawing viability, and remained viable for up to 6 days of in vitro culture. (C) 2012 Elsevier Inc. All rights reserved.

Effects of extrusion, lipid concentration and purity on physico-chemical and biological properties of cationic liposomes for gene vaccine applications

We developed cationic liposomes containing DNA through a conventional process involving steps of (i) preformation of liposomes, (ii) extrusion, (iii) drying and rehydration and (iv) DNA complexation. Owing to its high prophylactic potentiality against tuberculosis, which had already been demonstrated in preclinical assays, we introduced modifications into the conventional process towards getting a simpler and more economical process for further scale-up. Elimination of the extrusion step, increasing the lipid concentration (from 16 to 64 mM) of the preformed liposomes and using good manufacturing practice bulk lipids (96-98% purity) instead of analytical grade purity lipids (99.9-100%) were the modifications studied. The differences in the physico-chemical properties, such as average diameter, zeta potential, melting point and morphology of the liposomes prepared through the modified process, were not as significant for the biological properties, such as DNA loading on the cationic liposomes, and effective immune response in mice after immunisation as the control liposomes prepared through the conventional process. Beneficially, the modified process increased productivity by 22% and reduced the cost of raw material by 75%.

Methods of body-mass reduction by combat sport athletes

The aim of this study was to investigate the methods adopted to reduce body mass (BM) in competitive athletes from the grappling (judo, jujitsu) and striking (karate and tae kwon do) combat sports in the state of Minas Gerais, Brazil. An exploratory methodology was employed through descriptive research, using a standardized questionnaire with objective questions self-administered to 580 athletes (25.0 +/- 3.7 yr, 74.5 +/- 9.7 kg, and 16.4% +/- 5.1% body fat). Regardless of the sport, 60% of the athletes reported using a method of rapid weight loss (RWL) through increased energy expenditure. Strikers tend to begin reducing BM during adolescence. Furthermore, 50% of the sample used saunas and plastic clothing, and only 26.1% received advice from a nutritionist. The authors conclude that a high percentage of athletes uses RWL methods. In addition, a high percentage of athletes uses unapproved or prohibited methods such as diuretics, saunas, and plastic clothing. The age at which combat sport athletes reduce BM for the first time is also worrying, especially among strikers.

Water balance and spatial distribution of an anuran community from Brazil

Terrestrial amphibians may dehydrate when exposed to low humidity, representing an important factor affecting spatial distribution and community composition. In this study we investigated whether rates of dehydration and rehydration are able to explain the spatial distribution of an anuran community in a Restinga environment at the northern coast of the State of Bahia, Brazil, represented by 11 species distributed in 27 sample units. The environmental data set containing 20 variables was reduced to a few synthetic axes by principal component analysis (PCA). Physiological variables measured were rates of dehydration, rehydration from water, and rehydration from a neutral substrate. Multiple regression analyses were used to test the null hypothesis of no association between the environmental data set (synthetic axes of PCA) and each axis representative of a physiological variable, which was rejected (P < 0.001). Of 15 possible partial regressions only rehydration rate from neutral substrate vs. PC1. and PC2, rehydration rate from water vs. PC1, and dehydration rate vs. PC2 were significant. Our analysis was influenced by a gradient between two different groups of sample units: a beach area with high density of bromeliads and an environment without bodies of water with low density of bromeliads. Species of very specific natural history and morphological characters occur in these environments: Phyllodytes melanomystax and Scinax auratus, species frequently occurring in terrestrial bromeliads, and Ischnocnema paulodutrai, common along the northern coast of Bahia and usually found in forest remnants within environments with low number of bodies of water. In dry environments species with lower rates of dehydration were dominant, whereas species showing greater rates of dehydration were found predominantly in microhabitats with greater moisture or abundance of bodies of water.

FTIR and SEM analysis of CO2 laser irradiated human enamel

Objectives: Considering the enamel chemical structure, especially carbonate band, which has a major role in the caries prevention, the objective of the present study was to assess the chemical alterations on the enamel irradiated with CO2 laser by means of FTIR spectroscopy and SEM analysis. Design: The enamel surfaces were analysed on a spectrometer for acquisition of the absorption spectrum relative to the chemical composition of the control sample. The irradiation was conducted with a 10.6-mu m CO2 laser (0.55 W, 660 W/cm(2)). The carbonate absorption band at 1600-1291 cm(-1) as well as the water absorption band at 3793-2652 cm(-1) was measured in each sample after the irradiation. The water band was measured again 24-h after the irradiation. The band area of each chemical compound was delimited, the background was subtracted, and the area under each band was integrated. Each area was normalized by the phosphate band (1190-702 cm(-1)). Results: There was a statistically significant decrease (p < 0.05) in the water content after irradiation (control: 0.184 +/- 0.04; irradiated: 0.078 +/- 0.026), which increased again after rehydration (0.145 +/- 0.038). The carbonate/phosphate ratio was measured initially (0.112 +/- 0.029) and its reduction after irradiation indicated the carbonate loss (0.088 +/- 0.014) (p < 0.05). Conclusion: The 10.6-mu m CO2 laser irradiation diminishes the carbonate and water contents in the enamel after irradiation. (C) 2012 Elsevier Ltd. All rights reserved.

The need of a weight management control program in judo: a proposal based on the successful case of wrestling

Judo competitions are divided into weight classes. However, most athletes reduce their body weight in a few days before competition in order to obtain a competitive advantage over lighter opponents. To achieve fast weight reduction, athletes use a number of aggressive nutritional strategies so many of them place themselves at a high health-injury risk. In collegiate wrestling, a similar problem has been observed and three wrestlers died in 1997 due to rapid weight loss regimes. After these deaths, the National Collegiate Athletic Association had implemented a successful weight management program which was proven to improve weight management behavior. No similar program has ever been discussed by judo federations even though judo competitors present a comparable inappropriate pattern of weight control. In view of this, the basis for a weight control program is provided in this manuscript, as follows: competition should begin within 1 hour after weigh-in, at the latest; each athlete is allowed to be weighed-in only once; rapid weight loss as well as artificial rehydration (i.e., saline infusion) methods are prohibited during the entire competition day; athletes should pass the hydration test to get their weigh-in validated; an individual minimum competitive weight (male athletes competing at no less than 7% and females at no less than 12% of body fat) should be determined at the beginning of each season; athletes are not allowed to compete in any weight class that requires weight reductions greater than 1.5% of body weight per week. In parallel, educational programs should aim at increasing the athletes', coaches' and parents' awareness about the risks of aggressive nutritional strategies as well as healthier ways to properly manage body weight.