Modeling reticular and ventral ruminal pH of lactating dairy cows using ingestion and rumination behavior.

The prevention and control of metabolic and digestive diseases is an enormous challenge in dairy farming. Subacute ruminal acidosis (SARA) is assumed to be the most severe feed-related disorder and it impairs both animal health and economic efficiency. Currently, ruminal pH as well as variables derived from the daily pH curve are the main indicators for SARA. The objective of this study was to explain the daily pH course in the ventral rumen and reticulum of dairy cows using ingestion pattern and rumination behavior data gathered by automated data recording systems. The data of 13 ruminally fistulated lactating cows were collected at the experimental station of the Friedrich-Loeffler-Institut (Brunswick, Germany). The data included continuous pH measurements, which were recorded simultaneously in the reticulum by pH-measuring boluses and in the ventral rumen by a separate data logger. In addition, rumination behavior was measured using jaw movement sensors, and feed and water intakes were recorded by transponder-assisted systems. Milk yield and body weight were determined during and after each milking, respectively. For statistical evaluation, the data were analyzed using time-series modeling with multiple linear mixed regressions. Before applying the developed mathematical statistical modeling, we performed a plausibility assessment to ensure data quality. The major part of the mathematical statistical modeling consisted of data preparation, where all variables were transformed into a uniform 1-min resolution. Signal transformations were used to model individual feed and water intakes as well as rumination behavior events over time. Our results indicated that diurnal pH curves of both the reticulum and ventral rumen could be predicted by the transformed feed and water intake rates. Rumination events were associated with a marginal temporal increase in pH. We observed that the pH of the ventral rumen was delayed by approximately 37 min compared with that of the reticulum, which was therefore considered in the modeling. With the models developed in this study, 67.0% of the variance of the reticular pH curves and 37.8% of the variance of the ruminal pH curves could be explained by fixed effects. We deduced that the diurnal pH course is, to a large extent, associated with the animal's individual feed intake and rumination behavior.

Inter- and intra-observer reliability of animal welfare indicators for the on-farm self-assessment of fattening pigs.

In Germany all keepers of livestock are legally required to record animal welfare indicators as part of their on-farm self-assessment. The Association for Technology and Structures in Agriculture (Kuratorium für Technik und Bauwesen in der Landwirtschaft e.V. (KTBL)) has suggested the use of a particular set of animal welfare indicators in their publication Animal welfare indicators: Practical guide - Pigs. The aim of the present study was to evaluate the inter-observer reliability (Inter-OR) and intra-observer reliability (Intra-OR) of these indicators with respect to the welfare of fattening pigs. For the assessment of Inter-OR, three observers evaluated six KTBL animal welfare indicators. The Inter-OR of the indicators was calculated from the results using intra-class correlation coefficients (ICC). 'Excellent' Inter-OR results were found for the indicators tail length (ICC 0.89), skin lesions (ICC 0.77) and ear lesions (ICC 0.80). In contrast, the Inter-OR of the indicators tail lesions (ICC 0.46) and faecal soiling (ICC 0.47) were considered to be only 'fair' and that of the indicator lameness (ICC 0.36) as 'poor'. For the evaluation of the Intra-OR, the same three observers assessed the welfare of 162 to 200 fattening pigs using the same welfare indicators in total eight times. Again ICCs, here per indicator and observer, were used to calculate the Intra-OR. The Intra-OR of the indicators faecal soiling (ICC 0.81) and ear lesions (ICC 0.97) lay in the 'excellent' range on average. While the Intra-OR of the indicators skin lesions (ICC 0.67), tail length (ICC 0.74) and lameness (ICC 0.60) could still be considered as being 'good', the Intra-OR of the indicator tail lesions (ICC 0.52) could only be assessed as being 'fair'. From these results the significance of the KTBL indicators could be judged as follows: it is possible to use all the chosen indicators apart from the indicator tail lesions as an internal controlling instrument or as part of an internal weak-point analysis as long as the indicators are evaluated by the same person. A comparison of the indicators tail lesions, lameness and faecal soiling when assessed by different observers should be considered critically because the Inter-OR of these three indicators could only be considered as being 'poor' to 'fair'.

Genomewide study and validation of markers associated with production traits in German Landrace boars.

We present results from a genomewide association study (GWAS) and a single-marker association study. The GWAS was performed with the Illumina PorcineSNP60 BeadChip from which 5 markers were selected for a validation analysis. Genetic effects were estimated for feed intake, weight gain, and traits of fat and muscle tissue in German Landrace boars kept on performance test stations. The GWAS was performed in a population of 288 boars and the validation study for another 432 boars. No statistically significant effect was found in the GWAS after adjusting for multiple testing. Effects of 2 markers, which were significant genomewide before correction for multiple testing (P < 0.00005), could be confirmed in the validation study. The major allele of marker ALGA0056781 on SSC1 was positively associated with both higher weight gain and fat deposition. The effect on live-weight gain was 2.25 g/d in the GWAS (P = 0.0003) and 3.73 g/d in the validation study (P = 0.01) and for back fat thickness was 0.15 mm in the GWAS (P < 0.0001) and 0.20 mm in the validation study (P = 0.02). The marker had similar effects on test-day weight gain (GWAS: 3.85 g/d, P = 0.001; validation study: 6.80 g/d, P = 0.003) and back fat area (GWAS: 0.27 cm(2), P < 0.0001; validation study: 0.35 cm(2), P = 0.03). Marker ASGA0056782 on SSC13 was associated with live-weight gain. The major allele had negative effects in both studies (GWAS: -4.88 g/d, P < 0.0001; validation study: -3.75 g/d, P = 0.02). The effects of these 2 markers would have been excluded based on the GWAS alone but were shown to be significantly trait associated in the validation study indicating a false-negative result. The G protein-coupled receptor 126 (GPR126) gene approximately 200 kb downstream of marker ALGA0001781 was shown to be associated with human height and therefore might explain the association with weight gain in pigs. Several traits were affected in an economically desired direction by the minor allele of the markers, pointing to the possibility of improvement through further selection.

Relationship between hair fatty acid profile, reproduction, and milk performance in Holstein Friesian cows.

The aim of this study was to analyze the relationship between the fatty acid profiles of hair of first parity cows and their reproduction and milk performance. Hair samples from 46 German Holstein cows, 50 to 62 d postpartum, were analyzed to assess the relationship. All cows were fed the same diet. The lipid composition of hair was determined by gas chromatography. The fat content in the hair was 0.86%. Hair lipids contributed 75.14% SFA, 15.81% MUFA, and 9.92% PUFA. Cows were classified into a high and a low performance group according to their a) length of the interval from calving to first insemination (CFI), b) 100-d protein yield (100-d PY), and c) length of the interval from calving to conception (CC) and 100-d PY. Cows with short CFI or high 100-d PY or short CC and high 100-d PY had significantly greater proportions of de novo synthesized C12:0 (P < 0.05) compared with the corresponding low performing groups. Increased proportions of C12:0 in the hair were associated with shorter CFI (r = -0.32, P = 0.033) and greater 100-d PY (r = 0.34, P = 0.021). Furthermore, a discriminant analysis with C12:0 as a variable best predicted short CC and high 100-d PY (error rate 33.5%). Greater proportions of de novo synthesized fatty acids in the hair, in particular C12:0, are likely to reflect a sufficient energy availability that is required for fatty acid synthesis in ruminants.

Early and late onset of voluntary exercise have differential effects on the metabolic syndrome in an obese mouse model.

In a mouse model for juvenile obesity, we investigated how the age of onset of voluntary exercise affects factors of the metabolic syndrome. One exercise group had access to running wheels from 3 weeks (representing childhood) and another one from 9 weeks on (early adulthood). Both groups were compared to mice without exercise. The investigations were performed under 2 diets (standard maintenance and high-fat diet). Average daily running activity was independent of diet and exercise. On both diets, mice with exercise from 3 weeks on gained 10 g body weight and 5 g fat mass less than mice without exercise. The highest body weight difference between mice on HFD without exercise and mice on standard maintenance diet with exercise was 24 g. Despite the higher energy expenditure during exercise, young mice did not increase their energy intake adjusted for lean mass, while mice with exercise from 9 weeks had an increased energy intake of 6 kJ per day and therefore could not reduce fat mass on both diets. However, mice with exercise from 9 weeks had better glucose tolerance at 20 weeks than mice with exercise from childhood on. Independently of the age of exercise onset, triglycerides were reduced from 2.4 to 1.7 mmol/l on both diets and insulin levels from 1.5 to 0.3 and 4.5 to 1.8 µg/ml on standard maintenance and high-fat diet, respectively, which represents a considerable improvement. Physical activity seems to have long-lasting effects on body composition and health, but they are different depending on when exercise has begun.

A unique genetic defect on chromosome 3 is responsible for juvenile obesity in the Berlin Fat Mouse.

OBJECTIVE: This study aimed at the mapping and estimation of genetic and sex effects contributing to the obese phenotype of the Berlin Fat Mouse Inbred line 860 (BFMI860). This mouse line is predisposed for juvenile obesity. BFMI860 mice accumulate 24% total fat mass at 10 weeks of age under a standard maintenance diet. DESIGN: A total of 471 mice of a (BFMI860 x C57BL/6NCrl) F2 intercross population were fed a standard maintenance diet and were analysed for body composition at 10 weeks when they finished their rapid growth phase. RESULTS: The most striking result was the identification of a novel obesity locus on chromosome 3 (Chr 3) at 40 Mb, explaining 39% of the variance of total fat mass in the F2 population under a standard diet. This locus was named jObes1 (juvenile obesity 1). The BFMI860 allele effect was recessive. Males and females homozygous at jObes1 had on average 3.0 and 3.3 g more total fat mass at 10 weeks than the other two genotype classes, respectively. The effect was evident in all white adipose tissues, brown adipose tissue and also in liver. The position of the Chr 3 effect is syntenic to an obesity locus in humans. Additional loci for total fat mass and different white adipose tissue weights with minor effects were detected on mouse Chr 5 and 6. Another locus on Chr 4 had influence especially on liver weight. Many loci including jObes1 affected males and females to a different extent. CONCLUSION: The major locus on Chr 3 for juvenile obesity and its interaction with sex is unique and makes the BFMI860 mice an interesting resource for the discovery of novel genetic factors predisposing obesity, which might also contribute to obesity in humans. The results suggested that metabolic and regulatory pathways differed between the sexes.

Information theoretical probe selection for hybridisation experiments.

MOTIVATION: The choice of probes is an important feature of hybridisation experiments. In this paper we present an algorithm that optimises probes with respect to a training set of sequences based on Shannon entropy as a quality criterion. The practical motivation for our algorithm is oligonucleotide fingerprinting, a method for the simultaneous identification of sequences (cDNA or genomic DNA) by their hybridisation tags according to a set of short probes such as octamers, although the algorithm is of course not restricted to that application. RESULTS: We can show that our method is superior to the selection of probes according to their frequencies, which is a widely used strategy, and to randomly chosen probe sets. The quality of probe sets is assessed by a simulation pipeline that entails the set of probes as a simulation parameter. The performance of probe sets trained on sequences from different organisms shows additionally that probes should be chosen with regard to the organism under analysis. Case studies are presented on how constraints (G+C-content, complexity of the individual probes) influence the selection process. AVAILABILITY: A description of the oligonucleotide fingerprinting pipeline is published on our web-page http://www.molgen.mpg.de/ approximately ag_onf/met.htm. An executable of the algorithm and probe lists designed for human and rodents can be downloaded from the ftp-site ftp://ftp.molgen.mpg.de/pub/mpimg/probe_design/.

An algorithm for clustering cDNA fingerprints.

Clustering large data sets is a central challenge in gene expression analysis. The hybridization of synthetic oligonucleotides to arrayed cDNAs yields a fingerprint for each cDNA clone. Cluster analysis of these fingerprints can identify clones corresponding to the same gene. We have developed a novel algorithm for cluster analysis that is based on graph theoretic techniques. Unlike other methods, it does not assume that the clusters are hierarchically structured and does not require prior knowledge on the number of clusters. In tests with simulated libraries the algorithm outperformed the Greedy method and demonstrated high speed and robustness to high error rate. Good solution quality was also obtained in a blind test on real cDNA fingerprints.

Differential gene expression by endothelial cells in distinct angiogenic states.

Angiogenesis is a complex process that can be regarded as a series of sequential events comprising a variety of tissue cells. The major problem when studying angiogenesis in vitro is the lack of a model system mimicking the various aspects of the process in vivo. In this study we have used two in vitro models, each representing different and distinct aspects of angiogenesis. Differentially expressed genes in the two culture forms were identified using the suppression subtractive hybridization technique to prepare subtracted cDNA libraries. This was followed by a differential hybridization screen to pick up overexpressed clones. Using comparative multiplex RT-PCR we confirmed the differential expression and showed differences up to 14-fold. We identified a broad range of genes already known to play an important role during angiogenesis like Flt1 or TIE2. Furthermore several known genes are put into the context of endothelial cell differentiation, which up to now have not been described as being relevant to angiogenesis, like NrCAM, Claudin14, BMP-6, PEA-15 and PINCH. With ADAMTS4 and hADAMTS1/METH-1 we further extended the set of matrix metalloproteases expressed and regulated by endothelial cells.

Exhaustive mining of EST libraries for genes differentially expressed in normal and tumour tissues.

A four-step procedure for the efficient and systematic mining of whole EST libraries for differentially expressed genes is presented. After eliminating redundant entries from the EST library under investigation (step 1), contigs of maximal length are built upon each remaining EST using about 4 000 000 public and proprietary ESTs (step 2). These putative genes are compared against a database comprising ESTs from 16 different tissues (both normal and tumour affected) to determine whether or not they are differentially expressed (step 3; electronic northern). Fisher's exact test is used to assess the significance of differential expression. In step 4, an attempt is made to characterise the contigs obtained in the assembly through database comparison. A case study of the CGAP library NCI_CGAP_Br1.1, a library made from three (well, moderately, and poorly differentiated) invasive ductal breast tumours (2126 ESTs in total) was carried out. Of the maximal contigs, 139 were found to be significantly (alpha = 0.05) over-expressed in breast tumour tissue, while 13 appeared to be down-regulated.

Microarrays--chances and challenges.

Microarrays are a powerful tool in modern genome analysis. The massive parallel analysis of the RNA expression level of thousands of genes accelerates the research in molecular biology and leads to new insights in signalling pathways. Furthermore, genomic gains or losses can be analyzed for complete genomes in a single experiment. This creates new opportunities for understanding distinct chromosomal changes in the development of cancer. The ability to genotype patients using single nucleotide polymorphisms is of primordial interest to the pharmaceutical industry and may result in a more individualized medicine. Tissue microarrays are very useful in the investigation of the expression of a particular gene in hundreds of specimens by in situ hybridization. Also, protein microarrays are currently being developed for studying protein-protein interactions. In conclusion, microarrays will revolutionize all aspects of molecular biology, and will probably have the same impact as the polymerase chain reaction.

Estimating the entropy of DNA sequences.

The Shannon entropy is a standard measure for the order state of symbol sequences, such as, for example, DNA sequences. In order to incorporate correlations between symbols, the entropy of n-mers (consecutive strands of n symbols) has to be determined. Here, an assay is presented to estimate such higher order entropies (block entropies) for DNA sequences when the actual number of observations is small compared with the number of possible outcomes. The n-mer probability distribution underlying the dynamical process is reconstructed using elementary statistical principles: The theorem of asymptotic equi-distribution and the Maximum Entropy Principle. Constraints are set to force the constructed distributions to adopt features which are characteristic for the real probability distribution. From the many solutions compatible with these constraints the one with the highest entropy is the most likely one according to the Maximum Entropy Principle. An algorithm performing this procedure is expounded. It is tested by applying it to various DNA model sequences whose exact entropies are known. Finally, results for a real DNA sequence, the complete genome of the Epstein Barr virus, are presented and compared with those of other information carriers (texts, computer source code, music). It seems as if DNA sequences possess much more freedom in the combination of the symbols of their alphabet than written language or computer source codes.

The modular structure of informational sequences.

It is shown that DNA sequences can be decomposed into smaller units much the same as texts can be decomposed into syllables, words, or groups of words. Those smaller units (modules) are extracted from DNA sequences according to statistical criteria. Tests with sequences of known modular structure (two novels and a FORTRAN source code) were performed. The rate to which DNA sequences can be decomposed into modules (modularity) turns out to be a very sensitive measure to distinguish DNA sequences from random sequences.