Survey of disbudding practice on Czech dairy farms.

Disbudding is a routine practice in many dairy herds due to the effort to decrease the risk of injuries. Although the disbudding practice is regulated, it can vary among farms. The variation may be caused by many factors, such as herd size or type of breed, but also by farmers' perception of pain caused by disbudding. Hence, the aim of this study was to specify the disbudding practice on dairy farms in the Czech Republic and to assess these practices, including the use of pain mitigation medication, by breed, herd size, and sex of the disbudded calves. We analyzed data from 106 Czech dairy farms, which were collected by a trained interviewer at dairy meetings in 2014-2015. The farmers answered questions regarding the farm's basic characteristics, disbudding practice, and his/her attitude to the pain caused by disbudding. To test the influence of breed, herd size, and sex of disbudded calves on different variables, logistic regression models were used. Disbudding was performed in 92.5% of the surveyed farms; 63.3% of dairy calves were disbudded before 4 wk of age, and Czech Fleckvieh calves were 2.8 times more likely to be disbudded before 4 wk of age than Holstein calves. The hot-iron method was the most used method (69.4%). Calves were 4.5 times more likely to be disbudded by hot iron in herds where both heifers and bulls were disbudded than in herds where only heifers were disbudded. Most (>90%) surveyed farms did not use any pre- or post-procedure medication to mitigate the pain caused by disbudding. The disbudding procedure was performed mainly by farm personnel (94.9%), who were trained by a veterinarian or veterinary technician (46.9%) or by other farm personnel (37.8%) or were not trained (15.3%). Two-thirds of farmers observed behavioral changes in calves after disbudding. Most farmers estimated the pain caused by disbudding to be mild or moderate (20.4 or 45.9%, respectively) and 15.3% of farmers estimated it to be severe. Almost a quarter of interviewed farmers were unable to assess the duration of pain, 39.8% farmers estimated that the pain lasts only several minutes, and 20.4% estimated that the pain lasts up to 6 h. We detected a tendency that farmers of larger herds estimated pain duration to be shorter (odds ratio = 1.2). To improve calves' welfare, training is needed in disbudding practice, focusing on the advantage of pain mitigation.

The detection of proviral DNA by semi-nested polymerase chain reaction and phylogenetic analysis of Czech Maedi-Visna isolates based on gag gene sequences.

A semi-nested polymerase chain reaction (snPCR) for detecting proviral DNA of ovine lentivirus (OvLV) in peripheral blood mononuclear cells was developed. Primers for snPCR were situated within the gag gene of the Maedi-Visna virus (MVV) genome. A comparison between the snPCR and serological tests (agar gel immunodiffusion test, immunoblot) were performed using 98 ovine blood samples. Thirty (30.6%) of the 98 sheep examined had antibodies specific for the MVV. PCR showed 21 of them to be positive and nine seropositive animals to be PCR negative. Six of the 68 serologically negative sheep were found to be PCR positive, probably due to delayed seroconversion. The PCR amplification products of these six sheep were sequenced and subjected to phylogenetic analysis. The resulting phylogenetic tree of partial gag gene sequences confirmed that the ovine lentivirus genotype in the Czech Republic is more closely related to the prototype MVV isolates than to the caprine arthritis encephalitis viruses.

The role of cysteine residues in structure and enzyme activity of a maize beta-glucosidase.

The maize Zm-p60.1 gene encodes a beta-glucosidase that can release active cytokinins from their storage forms, cytokinin-O-glucosides. Mature catalytically active Zm-p60.1 is a homodimer containing five cysteine residues per a subunit. Their role was studied by mutating them to alanine (A), serine (S), arginine (R) or aspartic acid (D) using site-directed mutagenesis, and subsequent heterologous expression in Escherichia coli. All substitutions of C205 and C211 resulted in decreased formation and/or stability of the homodimer, manifested as accumulation of high levels of monomer in the bacterial expression system. Examination of urea- and glutathione-induced dissociation patterns of the homodimer to the monomers, HPLC profiles of hydrolytic fragments of reduced and oxidized forms, and a homology-based three-dimensional structural model revealed that an intramolecular disulfide bridge formed between C205 and C211 within the subunits stabilized the quaternary structure of the enzyme. Mutating C52 to R produced a monomeric enzyme protein, too. No detectable effects on homodimer formation were apparent in C170 and C479 mutants. Given the Km values for C170A/S mutants were equal to that for the wild-type enzyme, C170 cannot participate in enzyme-substrate interactions. Possible indirect effects of C170A/S mutations on catalytic activity of the enzyme were inferred from slight decreases in the apparent catalytic activity, k'cat. C170 is located on a hydrophobic side of an alpha-helix packed against hydrophobic amino-acid residues of beta-strand 4, indicating participation of C170 in stabilization of a (beta/alpha)8 barrel structure in the enzyme. In C479A/D/R/S mutants, Km and k'cat were influenced more significantly suggesting a role for C479 in enzyme catalytic action.

A relationship between K-ras gene mutations and some clinical and histologic variables in patients with primary colorectal carcinoma.

Mutations in the Kirsten ras 2 (K-ras) gene were described as early events in the process of colorectal carcinogenesis. The aim of this study was to find a possible relationship between the presence of K-ras mutation in samples of primary colorectal carcinomas and the clinico-pathological data of the investigated patients. Mutation in codon 12 of the K-ras gene was determined in 18 of 53 colorectal carcinomas (34%) in our group of patients. The presence of K-ras gene mutations was not related to gender, age of subject at diagnosis, staging or cancer location (p > 0.05). Sixteen of the 42 (38%) moderately differentiated carcinomas, and two of the eight (25%) well differentiated carcinomas contained K-ras mutation in codon 12, but none of the three poorly differentiated carcinomas contained the mutation. Moderately differentiated tumours contained an aspartate code GAT (in eight cases), a valine code GTT (in six cases), an alanine code GCT (in one case) and a serine code AGT (in one case) in codon 12. Well differentiated tumours contained only the valine code GTT (two cases). Our results show that the frequency of mutations in the K-ras gene in carcinomas in Central Europe is not different from the frequencies found in other parts of the world. The homogeneous incidence of K-ras mutation does not seem to be related to ethnic factors, dietary habits, or the composition of the diet.