Improved assessment of frozen/thawed mouse spermatozoa using fluorescence microscopy

Genetically modified (GM) animals are unique mutants with an enormous scientific potential. Cryopreservation of pre-implantation embryos or spermatozoa is a common approach for protecting these lines from being lost or to store them in a repository. A mutant line can be taken out of a breeding nucleus only if sufficient numbers of samples with an appropriate level of quality are cryopreserved. The quality of different donors within the same mouse line might be heterogeneous and the cryopreservation procedure might also be error-prone. However, only limited amounts of material are available for analysis. To improve the monitoring of frozen/thawed spermatozoa, commonly used in vitro fertilization (IVF) followed by embryo transfer were replaced with animal-free techniques. Major factors for assessing spermatozoa quality (i.e., density, viability, motility, and morphology) were evaluated by fluorescence microscopy. For this, a live/dead cell staining protocol requiring only small amounts of material was created. Membrane integrity was then examined as major parameter closely correlated with successful IVF. These complex analyses allow us to monitor frozen/thawed spermatozoa from GM mice using a relatively simple staining procedure. This approach leads to a reduction of animal experiments and contributes to the 3R principles (replacement, reduction and refinement of animal experiments).     

Experimentally induced cholangiohepatopathy by dosing sheep with fractionated extracts from Brachiaria decumbens.

Cholangiohepatopathy was induced in 5 lambs by oral administration of extracts from signal grass (Brachiaria decumbens) in Brazil. Grossly there were pale foci multifocally distributed throughout the hepatic parenchyma in 4 lambs. The microscopic changes, which were similar to those produced by other steroidal sapogenins-containing plants such as Tribulus terrestris and Panicum spp., included multifocal cholangitis, bile duct proliferation, and the presence of crystals in the biliary system.     

Evaluation of antirotavirus activity of flavonoids

Flavonoids are dietary components and the most ubiquitous phenolic compounds found in nature, showing a range of pharmacological activities including antiviral action. This study describes the antiviral screening of 60 different flavones and flavonols against human rotavirus (Wa-1 strain) as well as their cytotoxicity in MA104 cells. Cytotoxicity was investigated by cell morphology assessment and antirotavirus activity by cytopathic effect inhibition. Results were expressed as CC₅₀ and IC₅₀, respectively, in order to calculate the selectivity index (SI = CC₅₀/IC₅₀) of each compound. Structure–activity relationships (SAR) were proposed based on antirotavirus activity.     

Estimation of genetic effects in the presence of multicollinearity in multibreed beef cattle evaluation

Breed additive, dominance, and epistatic loss effects are of concern in the genetic evaluation of a multibreed population. Multiple regression equations used for fitting these effects may show a high degree of multicollinearity among predictor variables. Typically, when strong linear relationships exist, the regression coefficients have large SE and are sensitive to changes in the data file and to the addition or deletion of variables in the model. Generalized ridge regression methods were applied to obtain stable estimates of direct and maternal breed additive, dominance, and epistatic loss effects in the presence of multicollinearity among predictor variables. Preweaning weight gains of beef calves in Ontario, Canada, from 1986 to 1999 were analyzed. The genetic model included fixed direct and maternal breed additive, dominance, and epistatic loss effects, fixed environmental effects of age of the calf, contemporary group, and age of the dam x sex of the calf, random additive direct and maternal genetic effects, and random maternal permanent environment effect. The degree and the nature of the multicollinearity were identified and ridge regression methods were used as an alternative to ordinary least squares (LS). Ridge parameters were obtained using two different objective methods: 1) generalized ridge estimator of Hoerl and Kennard (R1); and 2) bootstrap in combination with cross-validation (R2). Both ridge regression methods outperformed the LS estimator with respect to mean squared error of predictions (MSEP) and variance inflation factors (VIF) computed over 100 bootstrap samples. The MSEP of R1 and R2 were similar, and they were 3% less than the MSEP of LS. The average VIF of LS, R1, and R2 were equal to 26.81, 6.10, and 4.18, respectively. Ridge regression methods were particularly effective in decreasing the multicollinearity involving predictor variables of breed additive effects. Because of a high degree of confounding between estimates of maternal dominance and direct epistatic loss effects, it was not possible to compare the relative importance of these effects with a high level of confidence. The inclusion of epistatic loss effects in the additive-dominance model did not cause noticeable reranking of sires, dams, and calves based on across-breed EBV. More precise estimates of breed effects as a result of this study may result in more stable across-breed estimated breeding values over the years.     

Cloning and structural analysis of equine platelet endothelial cell adhesion molecule (PECAM, CD31) and vascular cell adhesion molecule-1 (VCAM-1, CD106)

Platelet endothelial cell adhesion molecule (PECAM, CD31) and vascular cell adhesion molecule-1 (VCAM-1, CD106) are essential for leukocyte emigration and diapedesis. PECAM is an essential histologic marker of endothelial cells; VCAM-1 is a prototype marker for endothelial cell activation. In this study, equine PECAM and VCAM mRNA were cloned and sequenced. Both genes are highly conserved amongst several species. This study also revealed conserved structural and regulatory motifs, emphasizing the importance of these genes' physiological roles in immunological responses.     

Association of a single nucleotide polymorphism in the calpastatin gene with carcass and meat quality traits of beef cattle

Calpastatin (CAST) is a naturally occurring protein that inhibits the normal tenderization of meat as it ages postmortem. A SNP was identified in the CAST gene (a G to C substitution) and genotyped on crossbred commercially fed heifers (n = 163), steers (n = 226), and bulls (n = 61) from beef feedlots, and steers (n = 178) from a University of Guelph feeding trial. The association of the CAST SNP with carcass and meat quality traits was studied. Carcass traits included fat, lean, and bone yield; grade fat; LM area; and HCW. Meat quality traits included marbling grade; i.m. fat content of LM; tenderness evaluation of LM (Warner-Bratzler shear force) at 2, 7, 14, and 21 d of postmortem aging; and tenderness evaluation of semitendinosus muscle at 7 d of postmortem aging. The mixed model used in the analyses included fixed effects of CAST genotype, sex, slaughter group, and breed composition (linear covariate); sire was a random effect. For the analysis of shear force, i.m. fat content of LM was also included in the model as a linear covariate. Shear force measures were analyzed within days of postmortem aging and by repeated measures analysis. The CAST SNP allele C was more frequent (63%) in the crossbred population than allele G. The CAST SNP was associated with shear force across days of postmortem aging (P = 0.005); genotype CC yielded beef that was more tender than GG (-0.32 kg +/- 0.13), and CG had intermediate tenderness. The corresponding average allele substitution effect (G to C substitution) was also highly significant (-0.15 +/- 0.05 kg, P = 0.002). A lower percentage of unacceptably tough steaks (shear force > 5.7 kg) at 2 and 7 d postmortem was associated with an increasing number of C alleles (P < or = 0.05). At 7 d postmortem, the percentage of unacceptably tough steaks decreased by 24 and 35%, respectively, for animals carrying 1 and 2 copies of the C allele relative to animals with no C alleles. However, genotype CC had a greater fat yield (+1.44 +/- 0.56%; P = 0.037) than genotype GG, with a corresponding allele substitution effect of 0.67 +/- 0.27% (P = 0.015). Therefore, the CAST SNP allele C was associated with increased LM tenderness across days of postmortem aging and, importantly for the beef industry, had a significant reduction in the percentage of steaks rated unacceptably tough by consumers based on an assumed threshold level.     

Cytotoxic Activity of Semi-Synthetic Derivatives of Elatol and Isoobtusol

In the present study, the in vitro cytotoxic effects of six semi-synthetic derivatives of elatol (1) and isoobtusol (2) were investigated. Chemical modifications were performed on the hydroxyl groups aiming to get derivatives of different polarity, namely the hemisuccinate, carbamate and sulfamate. The structural elucidation of the new derivatives was based on detailed NMR and MS spectroscopic analyses. The in vitro cytotoxicity of compounds 1 to 8 was evaluated against A459 and RD tumor cell lines with CC₅₀ values ranging from 4.93 to 41.53 µM. These results suggest that the structural modifications performed on both compounds could be considered a good strategy to obtain more active derivatives.     

Efficacy of maduramicin against ionophore-tolerant field isolates of coccidia in broilers

Maduramicin ammonium was given at 2.5-8 ppm in the feed to broilers experimentally infected with coccidia recently isolated from broiler farms where ionophores had been used for several years. Infection pressure varied from mild to severe in five trials: mortality in unmedicated controls ranged from 0 to 59%, intestinal lesion scores were high, and weight gain was depressed by the infections. The cultures of Eimeria were partly resistant to ionophores: birds medicated with monensin at 100-121 ppm had only modest reductions in lesion scores and incomplete protection against weight loss or mortality. Control of infections by maduramicin was significant at 4 ppm but best at 5-7 ppm. Maduramicin was more effective than monensin or narasin, but about the same as salinomycin, in reducing lesions and mortality and in protecting performance. Maduramicin was well tolerated within the dose range of 5-7 ppm.