A method for estimating the outer exposure of dairy cows to deoxynivalenol (DON) and zearalenone (ZEN) as a precondition for risk assessment based on inner exposure with toxin residue levels in blood and urine as indicators.

DON and ZEN residues in the blood and urine of dairy cows can be used to predict the outer exposure to DON and ZEN expressed per kilogram diet for a risk evaluation based on comparisons to critical dietary concentrations. This method was used to evaluate the exposure of dairy cows from 12 farms located in Brandenburg, Germany, fed rations with unknown DON and ZEN concentrations (N = 244). The corresponding diet concentrations predicted by different methods from analyzed blood and urine samples varied significantly amongst farms from 0 to 1.6 mg/kg for DON and 0 to 3.0 mg/kg for ZEN at a reference dry matter content of 88% but independently of lactational state (post-partum vs. early lactation). This significant variation was noticed below the critical dietary DON concentration of 5 mg/kg, while the ZEN concentration in one farm exceeded the critical ZEN level of 0.5 mg/kg markedly. Predicted DON concentrations of rations increased with the proportion of maize silage, while the high ZEN concentration found in one farm was most likely related to a higher proportion of sugar beet pulp supposedly highly contaminated by ZEN. Exceeding the critical dietary ZEN concentration and significant variations in DON contents below the critical level was not related to performance, reproductive performance, and health-related traits of cows. For a more consistent evaluation of possible associations between the inner exposure of cows to DON and ZEN, more frequent longitudinal observations of both mycotoxin residue levels and performance and health traits are required.

Evaluation of multifactorial digestive disorders in a dairy herd at different stages of lactation.

The manager of a large dairy herd (total confinement, total mixed ration feeding, 10130 kg milk sold per cow and year) requested a workup of a digestive disorder problem that had been ongoing for several years. The cows were at all stages of lactation, and the incidence proportion (events/100 cows at risk) of digestive disorders was approximately 5 to 10%. The clinical picture included an abnormal demeanor of varying severity and signs that differed among the cows. The latter included decreased milk production and physical activity, low body condition score, abnormal rumen motility and stratification, small intestinal dilatation, diarrhea, undigested fiber particles in the feces, fever and abdominal pain.The following approach was used to investigate the digestive disorder problem:1. The herd was assessed for production levels, housing requirements, feeding protocols and animal health management. The latter comprised evaluation of different animal scores, metabolic profile analysis, diagnosis of disease, culling records and slaughter data. The results revealed risk factors concerning the feeding and animal health monitoring, (e. g. in dry matter intake and silage quality management, disease detection and diagnosis in fresh cows). The assessment also identified a high occurrence of digestive disorders of unknown origin.2. Fifteen cows that represented the ongoing digestive disorder problem were selected to undergo clinical examination, hematological analysis, urinalysis, and ultrasonography of the ventral abdomen. The clinical examinations revealed different digestive disorders, which were mainly inflammatory in nature, in all the cows. Eight cows had localized reticuloperitonitis and 13 had left displaced abomasum with different degrees of displacement and adhesions between the abomasum and reticulum attributable to reticuloperitonitis.Our results revealed a multifactorial problem caused by several risk factors relating to animal health and feeding management protocols that resulted in different types of digestive disorders. The wires from damaged tires used to hold the pit silo tarps in place were identified as a possible traumatic cause of the reticuloperitonitis. Treatment, prevention and follow-up of the different conditions were discussed.

Functional Variants Associated With CMPK2 and in ASB16 Influence Bovine Digital Dermatitis.

Bovine digital dermatitis (BDD) is an infectious disease of the hoof in cattle with multifactorial etiology and a polygenic influence on susceptibility. With our study, we identified genomic regions with the impact on occurrence and development of BDD. We used 5,040 genotyped animals with phenotype information based on the M-stage system for genome-wide association. Significant associations for single-nucleotide polymorphisms were found near genes CMPK2 (chromosome 11) and ASB16 (chromosome 19) both being implicated in immunological processes. A sequence analysis of the chromosomal regions revealed rs208894039 and rs109521151 polymorphisms as having significant influence on susceptibility to the disease. Specific genotypes were significantly more likely to be affected by BDD and developed chronic lesions. Our study provides an insight into the genomic background for a genetic predisposition related to the pathogenesis of BDD. Results might be implemented in cattle-breeding programs and could pave the way for the establishment of a BDD prescreening test.

Behavioral Observation Procedures and Tests to Characterize the Suitability of Sows for Loose-Housed Farrowing Systems.

The objective of the study was to evaluate behavioral observation procedures and tests to characterize sows' behavior for their suitability for free farrowing systems. Nest building activity (NB), lying-down behavior (LDB), and position after lying down (PLD) were assessed. Four tests were designed to characterize the reaction of sows to a novel object and an unexpected situation (Towel Test, TT), behavior towards humans (Dummy Arm Test, DAT; Trough Cleaning Test, TCT), and behavior towards piglets (Reunion Test, RT). The study was performed on a nucleus farm in 37 batches including 771 purebred landrace sows housed in farrowing pens with short-term fixation. The assessment of NB started 2 days before the expected date of the farrowing. In 56.2% of the observations, the sows showed increased chewing activity on gunnysacks. The LDB and PLD were assessed on days 3 and 19 post partum (p.p.). In 49.1% of the observations, sows showed careful lying-down behavior. In 50.1% of cases, sows preferred the stomach-teats-position when lying down. With the DAT on day 4 p.p., in 89.3% of observations, no or only slight reactions of the sow were documented. The TT and TCT were performed on days 3 and 10 p.p. Strong defensive reactions of animals towards humans were recorded in 4.5% of the observations in the TT, and in 4.0% of the observations in the TCT. In the RT on day 3 p.p., in 61.8%, a joyful response of the sows to the reunion with their piglets was observed. This study showed that the behavioral observation procedures and designed tests are suitable to characterize sows' behavior towards humans and piglets with regard to traits that are particularly important in systems without fixation.

Interdigital Hyperplasia in Holstein Cattle Is Associated With a Missense Mutation in the Signal Peptide Region of the Tyrosine-Protein Kinase Transmembrane Receptor Gene.

Bovine interdigital hyperplasia (IH) is a typical disease of the foot with varying prevalence depending on age, breed, and environmental factors resulting in different degrees of lameness. In studies based on assessments of claw health status at time of hoof trimming and applying genetic-statistical models to analyze this data, IH consistently exhibits high estimates of heritability in the range of 0.30-0.40. Although some studies have identified chromosomal regions that could possibly harbor causative genes, a clear identification of molecular causes for IH is lacking. While analyzing the large database of claw health status as documented at time of hoof trimming, we identified one herd with extreme prevalence of IH of > 50% of affected Holstein dairy cows. This herd subsequently was chosen as the object of a detailed study. A total of n = 91 cows was assessed and revealed a prevalence of 59.3% and 38.5% for IH cases, documented as "one-sided" or "two-sided", respectively. Cows were genotyped using the BovineSNP50 BeadChip. A genome wide association study revealed two significantly associated chromosomal positions (-log10P = 5.57) on bovine chromosome 8 (BTA8) located in intron 5 and downstream of the receptor tyrosine kinase-like orphan receptor 2 (ROR2) gene. As ROR2 plays a key role in ossification of the distal limbs and is associated with brachydactylies in humans, it was a reasonable candidate for IH. A comparative sequencing of the ROR2 gene between cases and controls revealed two missense variants in exon 1 (NC_037335.1:g.85,905,534T > A, ARS-UCD1.2) and exon 9 (NC_037335.1:g.86,140,379A > G, ARS-UCD1.2), respectively. Genotyping of both variants in the cohort of 91 cattle showed that the exon 1 variant (rs377953295) remained significantly associated with IH (p < 0.0001) as a risk factor of the disease. This variant resulted in an amino acid exchange (ENSBTAP00000053765.2:p.Trp9Arg) in the N-terminal region of the ROR2 signal peptide which is necessary for proper topology of the polypeptide during translocation. Quantification of ROR2 mRNA and ROR2 protein showed that the variant resulted in a significant suppression of ROR2 expression in homozygous affected compared to wild type and carrier cows.

Genetic analysis of new progesterone-based fertility traits in dairy cows measured on-farm.

The objectives of this study were (1) to analyze the agreement of a standard laboratory ELISA for progesterone (P4) with an automated on-farm ELISA kit operated under commercial conditions in 1,297 milk samples from 50 dairy cows; (2) to study the influence of the method of detection of luteal activity on genetic parameters of fertility traits based on P4 measured with an automated on-farm ELISA once weekly from wk 3 to 9 postpartum in the milk of 1,304 cows; and (3) to study the influence of sampling frequency (once or twice weekly from wk 3 to 9) on the same traits from 296 cows. Luteal activity can be detected when there is an active corpus luteum in the ovary producing P4 and indicating the onset of reproductive cyclicity after calving. The on-farm ELISA overestimated P4 contents by a mean square error of prediction of 2.76 ng/mL and had an intermediate Spearman correlation with the laboratory kit (0.54). For the second objective, the postpartum interval to the commencement of luteal activity (C-LA), proportion of luteal activity between d 15 and 63 postpartum (P-LA), calculated as the number of samples above the threshold for high P4 values divided by the number of all samples, and delay of first ovulation (DOV1), defined as C-LA occurring later than d 45 postpartum, were derived from the P4 profiles. Both C-LA and DOV1 were determined by (a) thorough qualitative visual inspection of the profile, (b) the profile's mean as threshold for the first increase in P4 postpartum, indicating commencement of luteal activity, and (c) 3 ng/mL as threshold for the first increase in P4, a value that has been used by many other studies. Similarly, P-LA was determined by using methods (b) and (c). Estimates of heritability were 0.04 to 0.13 for C-LA, 0.12 to 0.23 for P-LA, and 0.03 to 0.07 for DOV1. Genetic correlation of P-LA with C-LA and with the profile's mean P4 was -1.00. The profile's mean had a higher estimate of heritability (0.11-0.12) than C-LA or DOV1. It can be calculated as the arithmetic mean of all P4 values of a profile, whereas C-LA, P-LA, and DOV1 need a definition of a threshold for high P4 values. We therefore suggest the profile's mean as a promising candidate for further research. For the third objective, once-weekly sampling was mimicked by neglecting every second sample, and C-LA and DOV1 shifted toward a later onset of cyclicity. Thus, a common standard for sampling regimen and detection algorithm is essential to avoid incompatibility between studies.

Enlarging a training set for genomic selection by imputation of un-genotyped animals in populations of varying genetic architecture.

BACKGROUND: The most common application of imputation is to infer genotypes of a high-density panel of markers on animals that are genotyped for a low-density panel. However, the increase in accuracy of genomic predictions resulting from an increase in the number of markers tends to reach a plateau beyond a certain density. Another application of imputation is to increase the size of the training set with un-genotyped animals. This strategy can be particularly successful when a set of closely related individuals are genotyped. METHODS: Imputation on completely un-genotyped dams was performed using known genotypes from the sire of each dam, one offspring and the offspring's sire. Two methods were applied based on either allele or haplotype frequencies to infer genotypes at ambiguous loci. Results of these methods and of two available software packages were compared. Quality of imputation under different population structures was assessed. The impact of using imputed dams to enlarge training sets on the accuracy of genomic predictions was evaluated for different populations, heritabilities and sizes of training sets. RESULTS: Imputation accuracy ranged from 0.52 to 0.93 depending on the population structure and the method used. The method that used allele frequencies performed better than the method based on haplotype frequencies. Accuracy of imputation was higher for populations with higher levels of linkage disequilibrium and with larger proportions of markers with more extreme allele frequencies. Inclusion of imputed dams in the training set increased the accuracy of genomic predictions. Gains in accuracy ranged from close to zero to 37.14%, depending on the simulated scenario. Generally, the larger the accuracy already obtained with the genotyped training set, the lower the increase in accuracy achieved by adding imputed dams. CONCLUSIONS: Whenever a reference population resembling the family configuration considered here is available, imputation can be used to achieve an extra increase in accuracy of genomic predictions by enlarging the training set with completely un-genotyped dams. This strategy was shown to be particularly useful for populations with lower levels of linkage disequilibrium, for genomic selection on traits with low heritability, and for species or breeds for which the size of the reference population is limited.

Expression of microRNAs is not related to increased expression of ZDHHC9 in hind leg muscles of splay leg piglets.

ZDHHC9 (zinc finger, DHHC-type containing 9) is a protein acyl transferase involved in palmitoylation of basic signaling molecules. We found ZDHHC9 expression increased in hind leg muscles of newborn splay leg piglets. In order to elucidate the background of this increased expression we determined the structure of the porcine gene, including sequence variation, and analyzed the structure and expression of microRNAs potentially targeting the gene. We confirmed the expression results by RT Real-time PCR. The porcine ZDHHC9 gene has a similar structure to the human gene with two transcripts resulting in an identical protein. None of the 17 single nucleotide polymorphisms (SNPs) identified in the porcine gene affects the protein or putative microRNA binding sites, respectively. Two microRNAs (93 [minor] and 106b) were assayed in the muscles. Their expression variation proved to be independent from ZDHHC9 expression thus eliminating them as causally related to congenital splay leg.

Identification of candidate genes for congenital splay leg in piglets by alternative analysis of DNA microarray data.

The congenital splay leg syndrome in piglets is characterized by a temporarily impaired functionality of the hind leg muscles immediately after birth. Etiology and pathogenetic mechanisms for the disease are still not well understood. We compared genome wide gene expression of three hind leg muscles (M. adductores, M. gracilis and M. sartorius) between affected piglets and their healthy littermates with the GeneChip Porcine Genome Array (Affymetrix) in order to identify candidate genes for the disease. Data analysis with standard algorithms revealed no significant differences between both groups. By application of an alternative approach, we identified 63 transcripts with differences in two muscles and 5 genes differing between the groups in three muscles. The expression of six selected genes (SQSTM1, SSRP1, DDIT4, ENAH, MAF, and PDK4) was investigated with SYBRGreen RT-Real time PCR. The differences obtained with the microarray analysis could be confirmed and demonstrate the validity of the alternative approach to microarray data analysis. Four genes with different expression levels in at least two muscles (SQSTM1, SSRP1, DDIT4, and MAF) are assigned to transcriptional cascades related to cell death and may thus indicate pathways for further investigations on congenital splay leg in piglets.

Temporary consumption of diet with unbalanced amino acid pattern affects long-lasting growth retardation correlated with oxidative stress response associated gene expression in juvenile pigs.

BACKGROUND & AIMS: The present study was conducted to study whether oxidative stress is the trigger of long-term physiological effects of temporary consumption of a soy protein isolate (SPI) based diet with an imbalanced amino acid pattern. MATERIAL AND METHODS: Hepatic expression of 19 genes that are involved in and co-regulated with oxidative stress response and showing diet-associated expression after chronic SPI feeding using quantitative RT-PCR, growth and liver composition were investigated in a model of protein-underfeeding juvenile pigs, which were fed a casein (CAS) based diet for four weeks subsequent to a four week consumption of an SPI diet in comparison with chronically CAS fed animals. RESULTS: Temporary feeding of SPI diet resulted in prolonged up-regulation of genes involved in oxidative/cellular stress response (glutathione-S-transferase, peptide methionine sulfoxide reductase, calnexin, organic anion transport polypeptide 2). Cluster analysis of gene expression data indicated persistent SPI-related co-regulation of the genes involved in stress response with genes involved in the regulation of protein biosynthesis and in neuronal signalling for at least four weeks after replacement of SPI by CAS. Gene expression data are negatively correlated with body weight and liver protein content. CONCLUSION: Significant association of oxidative stress responsiveness with growth retardation and liver composition underline the possible impact of diet-affected oxidative stress for long-lasting deleteriously metabolic consequences.

Molecular cloning, mapping, and functional analysis of the bovine sulfate transporter SLC26a2 gene.

Sulfate is one of the most important macronutrients in cells and the major sulfur source in many organisms as well as one of the most abundant anions in the serum. As sulfate is a hydrophilic anion, movement across the lipid bilayer is mediated by transporters that regulate efflux and influx. Here, we report the molecular cloning, mapping, and functional analysis of the bovine solute carrier/sulfate transporter SLC26a2 gene, the first member of this family to be cloned in cattle. A recombinant phage library was screened, and single phages harbouring the SLC26a2 gene was isolated and sequenced. A fragment of 6295 base pairs (bp) of the bovine SLC26a2 gene harbouring exon 2 and exon 3 was used for further analysis. Similar to the human, ovine, mouse, and rat SLC26a2 gene, the bovine ortholog consists of two coding exons. The open reading frame harbours 2202 nucleotides (nt), coding for a protein of 734 amino acids with a calculated molecular weight of 81.5 kilodaltons (kDa) and a statistical isoelectric point (pI) of 8.77. The bovine SLC26a2 gene was mapped to chromosome 7q23-q24 (BTA 7q23-q24) by fluorescence in situ hybridisation (FISH) analysis. Two point mutations were identified comparing the DNAs of 300 Holstein Frisian cattle, one of them resulting in an isoleucine to serine amino acid exchange at position 520. The Ile520Ser exchange influences the sulfate uptake as measured in primary fibroblasts isolated from testis and in immortalized fibroblastoid bovine cell lines.

Dietary protein modifies hepatic gene expression associated with oxidative stress responsiveness in growing pigs.

Understanding the basis for differences in nutrient requirements and for nutrient effects on health and performance requires an appreciation of the links between nutrition and gene expression. We developed and applied molecular probes to characterize diet-associated postabsorptive hepatic gene expression in growing pigs chronically fed protein-restricted diets based on either casein (CAS) or soy protein isolate (SPI). Eighty-eight expressed sequence tags (ESTs) were identified on the basis of diet-related changes in expression, by using an mRNA differential display method. Expression profiling based on transcription analysis by real-time reverse transcriptase-polymerase chain reaction showed that the SPI diet significantly changed the pattern of gene expression as compared with the CAS diet and allowed identification of coregulated genes. The expression of six genes involved in the metabolism of stress response (glutathione S-transferase, peptide methionine sulfoxide reductase, apolipoprotein A-I, organic anion transport polypeptide 2, calnexin, heat shock transcription factor 1) exhibited significant changes in the transcription level and indicated an increased oxidative stress response in pigs fed the SPI diet. Hierarchical clustering of gene expression data of all 33 ESTs analyzed across 14 pigs fed the two different diets resulted in clustering of genes related to the oxidative stress response with genes related to the regulation of gene expression and neuronal signaling.