Cows selected for divergent mastitis susceptibility display a differential liver transcriptome profile after experimental Staphylococcus aureus mammary gland inoculation.

Infection and inflammation of the mammary gland, and especially prevention of mastitis, are still major challenges for the dairy industry. Different approaches have been tried to reduce the incidence of mastitis. Genetic selection of cows with lower susceptibility to mastitis promises sustainable success in this regard. Bos taurus autosome (BTA) 18, particularly the region between 43 and 59 Mb, harbors quantitative trait loci (QTL) for somatic cell score, a surrogate trait for mastitis susceptibility. Scrutinizing the molecular bases hereof, we challenged udders from half-sib heifers having inherited either favorable paternal haplotypes for somatic cell score (Q) or unfavorable haplotypes (q) with the Staphylococcus aureus pathogen. RNA sequencing was used for an in-depth analysis of challenge-related alterations in the hepatic transcriptome. Liver exerts highly relevant immune functions aside from being the key metabolic organ. Hence, a holistic approach focusing on the liver enabled us to identify challenge-related and genotype-dependent differentially expressed genes and underlying regulatory networks. In response to the S. aureus challenge, we found that heifers with Q haplotypes displayed more activated immune genes and pathways after S. aureus challenge compared with their q half-sibs. Furthermore, we found a significant enrichment of differentially expressed loci in the genomic target region on BTA18, suggesting the existence of a regionally acting regulatory element with effects on a variety of genes in this region.

Correction to: Characterization of functional traits with focus on udder health in heifers with divergent paternally inherited haplotypes on BTA18.

The original article [1] contained an error whereby the captions to Figs 2 and 3 were mistakenly inverted; this has now been corrected.

Characterization of functional traits with focus on udder health in heifers with divergent paternally inherited haplotypes on BTA18.

BACKGROUND: A major challenge in modern medicine and animal husbandry is the issue of antimicrobial resistance. One approach to solving this potential medical hazard is the selection of farm animals with less susceptibility to infectious diseases. Recent advances in functional genome analysis and quantitative genetics have opened the horizon to apply genetic marker information for efficiently identifying animals with preferential predisposition regarding health traits. The current study characterizes functional traits with a focus on udder health in dairy heifers. The animals were selected for having inherited alternative paternal haplotypes for a genomic region on Bos taurus chromosome (BTA) 18 genetically associated with divergent susceptibility to longevity and animal health, particularly mastitis. RESULTS: In the first weeks of lactation, the q heifers which had inherited the unfavorable (q) paternal haplotype displayed a significantly higher number of udder quarters with very low somatic cell count (< 10,000 cells / ml) compared to their paternal half-sib sisters with the favorable (Q) paternal haplotype. This might result in impaired mammary gland sentinel function towards invading pathogens. Furthermore, across the course of the first lactation, there was indication that q half-sib heifers showed higher somatic cell counts, a surrogate trait for udder health, in whole milkings compared to their paternal half-sib sisters with the favorable (Q) paternal haplotype. Moreover, heifers with the haplotype Q had a higher feed intake and higher milk yield compared to those with the q haplotype. Results of this study indicate that differences in milk production and calculated energy balance per se are not the main drivers of the genetically determined differences between the BTA18 Q and q groups of heifers. CONCLUSIONS: The paternally inherited haplotype from a targeted BTA18 genomic region affect somatic cell count in udder quarters during the early postpartum period and might also contribute to further aspects of animal's health and performance traits due to indirect effects on feed intake and metabolism.

Different milk diets have substantial effects on the jejunal mucosal immune system of pre-weaning calves, as demonstrated by whole transcriptome sequencing.

There is increasing evidence that nutrition during early mammalian life has a strong influence on health and performance in later life. However, there are conflicting data concerning the appropriate milk diet. This discrepancy particularly applies to ruminants, a group of mammals that switch from monogastric status to rumination during weaning. Little is known regarding how the whole genome expression pattern in the juvenile ruminant gut is affected by alternative milk diets. Thus, we performed a next-generation-sequencing-based holistic whole transcriptome analysis of the jejunum in male pre-weaned German Holstein calves fed diets with restricted or unlimited access to milk during the first 8 weeks of life. Both groups were provided hay and concentrate ad libitum. The analysis of jejunal mucosa samples collected 80 days after birth and four weeks after the end of the feeding regimes revealed 275 differentially expressed loci. While the differentially expressed loci comprised 67 genes encoding proteins relevant to metabolism or metabolic adaptation, the most distinct difference between the two groups was the consistently lower activation of the immune system in calves that experienced restricted milk access compared to calves fed milk ad libitum. In conclusion, different early life milk diets had significant prolonged effects on the intestinal immune system.

Mining long noncoding RNA in livestock.

Genome-wide association studies in livestock based on high-resolution genotyping and sequencing have revealed that the majority of signals associated with complex phenotypic traits are located outside of annotated protein-coding regions in the genome. The approaches of next-generation sequencing applied to whole transcriptome and chromatin profiles have provided information about existing genome-wide transcriptional activity and have revealed that the genomes are templates for thousands of long noncoding transcripts (lncRNAs). Despite their lack of coding capacity, many lncRNAs have been found to play functional roles in a variety of biological processes, which is adding a novel regulatory network to the complex structural organization and function of the genome. Here, we summarize main features of lncRNAs, provide an overview about computational tools and pipelines used for identification of lncRNAs from whole transcriptome datasets and review the current state of knowledge about lncRNAs in livestock species. Although lncRNAs are increasingly emerging as an integral component of the regulatory information encoded in the genome, the complexity of the transcriptomes in domesticated animals is inadequately characterized in comparison to human and mouse. Progress in elucidating whole transcriptomes of livestock species, including identification, functional annotation and characterization of lncRNAs, will be essential for a better understanding of basic biological processes associated with developmental, metabolic and immunological regulation and adaptation and phenotypic variation of complex traits in domesticated animals.

Vertebral and spinal dysplasia: A novel dominantly inherited congenital defect in Holstein cattle.

Monitoring and surveillance strategies are imperative for managing genetic defects in livestock populations in order to avoid detrimental effects on animal welfare and productivity. Recently, a number of previously unknown defects have been described in cattle, fostered by the huge progress in genome analysis and genomic selection. In response to reports about a potentially new defect in Holstein cattle, case-control studies were carried out to confirm a genetic background of the defect and to evaluate its phenotypic relevance. Eighty-five potentially affected offspring of a suspected carrier sire for the defect and 41 matched control calves were subjected to clinical and epidemiological monitoring on 39 farms. Forty-one animals, all offspring of the suspected carrier sire, showed pathognomonic tail malformations providing highly significant evidence for a congenital inherited defect, which was subsequently termed vertebral and spinal dysplasia (VSD). The defect is characterised by vertebral (specifically tail) deformities and neurological dysfunctions with gait abnormalities of the hind limbs. The deformities and neurological dysfunctions varied from very mild (only tail deformities) to severe (paraparesis). Detailed epidemiological monitoring provided no indication of environmental factors affecting VSD. The malformations and dysfunctions associated with VSD, as well as its mode of inheritance and the genotyping of the suspected carrier sire, indicated that VSD is a defect previously not described in cattle. VSD is inherited in a dominant mode, but shows incomplete penetrance of the phenotype, which impedes unequivocal identification of VSD carriers. A direct diagnostic genetic test for VSD is available.

Bovine neonatal pancytopenia (BNP): novel insights into the incidence, vaccination-associated epidemiological factors and a potential genetic predisposition for clinical and subclinical cases.

Bovine neonatal pancytopenia (BNP) is a haemorrhagic disease of newborn calves elicited by colostrum from specific cows. Two studies have indicated that BNP-inducing colostrum might be associated with alloantibodies directed against MHC class I in response to vaccination with a distinct inactivated viral vaccine. However, the proportion of alloantibody-producing individuals by far exceeds the proportion of clinical BNP cases in the vaccinated population. This raises the question about the incidence of subclinical, unrecognised cases and also suggests further factors involved in BNP pathogenesis, e.g., genetic predisposition. Our results on neonatal calves from a closely monitored resource population confirmed the hypothesis of a genetic predisposition for clinical BNP and suggest that the predisposition is also involved in subclinical BNP-cases. No indication was obtained for a higher frequency of subclinical BNP-cases compared with clinical cases. Neither time point nor frequency of vaccination was a relevant factor for BNP in our resource population.

Short communication: evaluation of bovine milk residues from routine milk testing programs as DNA source for genotyping.

Genome-wide association studies and genomic evaluation using a dense set of genetic markers both require a large number of genotyped individuals. Collection of the respective samples contributes substantially to the cost of the approach. In dairy cattle research, the use of residues from routine milk recording would be a cost-saving alternative to obtain samples for an appropriate number of individuals with specific phenotypes in a very short time. To assess the suitability of milk recording residues, we concurrently investigated milk residues obtained after standardized milk recording procedures and blood samples from 115 cows originating from 3 farms with different milking systems by genotyping 15 microsatellite markers. We found that 4% of the milk samples were possibly assigned to the wrong animal (i.e., conflicts) and that at least 27% of the milk residues were contaminated, as indicated by an extra allele not present in the blood sample. These additional alleles primarily originated from a sample with a higher somatic cell score that went through the milk sample analyzer in the milk laboratory before the target sample. Furthermore, additional allele carryover was observed across more than one sample, when the difference in somatic cell count between samples exceeded 100,000 cells/mL. Finally, in several samples, the extra allele could not be traced back to previous samples passing through the milk sample analyzer. One source of those contaminations might be sample collection on-farm due to milk traces from the previously milked cow in the hose. No correlation was found between the farm management and conflicts or contaminations. We conclude that residues from routine milk recording are not suitable for genomic evaluation or genome-wide association studies because of the high prevalence of contamination generated at several steps during the collection and processing of milk residual samples.

Tissue-specific mRNA expression patterns reveal a coordinated metabolic response associated with genetic selection for milk production in cows.

The molecular mechanisms regulating the physiological adaptation of tissues important for nutrient partitioning and metabolism in lactating cows are still not completely understood. The aim of our study was to identify tissue-specific regulatory mechanisms necessary to accommodate metabolic changes associated with different genetic potential for milk performance. For this purpose, we analyzed mRNA expression of genes involved in energy metabolism of segregating F(2) beef type cows with a combined genetic dairy and beef background (Charolais × German Holstein cross, CH×GH) in contrast to purebred German Holstein (GH) dairy cows. Three groups of cows differing in milk performance were examined using quantitative real-time PCR in liver, mammary gland, and skeletal muscle. Our results describe substantial tissue-specific differences in mRNA transcription profiles between cow groups in relation to their genetic potential for milk performance and highlight genes exhibiting specific, partially yet-unknown functions in dairy and beef type cows, e.g., upregulation of PCK2 transcripts in the mammary gland and FBP2 transcripts in skeletal muscle of dairy cows. Noticeably, PCCA and PPARGC1A mRNA abundance varied significantly across experimental groups in all three tissues, pointing to potential key gene functions in the metabolic adaptation relative to divergent milk production performance. Correlations of mRNA expression levels to milk performance traits indicate that gene transcriptional processes may play a regulatory role in liver, mammary gland, and skeletal muscle to enable cows with different genetic potential for milk performance to cope with metabolic lactation-associated challenges.

Revisiting the quantitative trait loci for milk production traits on BTA6.

A parallel association study was performed in two independent cattle populations based on 41 validated, targeted single nucleotide polymorphisms (SNPs) and four microsatellite markers to re-evaluate the multiple quantitative trait loci (QTL) architecture for milk performance on bovine chromosome 6 (BTA6). Two distinct QTL located in the vicinity of the middle region of BTA6, but differing unambiguously regarding their effects on milk composition and yield traits were validated in the German Holstein population. A highly significant association of the protein variant ABCG2 p.Tyr581Ser with milk composition traits reconfirmed the causative molecular relevance of the ABCG2 gene in QTL region 1, whereas in QTL region 2, significant and tentative associations between gene variants RW070 and RW023 (located in the promoter region and exon 9 of the PPARGC1A gene for milk yield traits) were detected. For the German Fleckvieh population, only RW023 showed a tentative association with milk yield traits, whereas those loci with significant effects in German Holsteins (ABCG2 p.Tyr581Ser, RW070) showed fixed alleles. Even though our new data highlight two variants in the PPARGC1A gene (RW023, RW070) in QTL region 2, based on the results of our study, currently no unequivocal conclusion about the causal background of this QTL affecting milk yield traits can be drawn. Notably, the German Holstein and Fleckvieh populations, known for their divergent degree of dairy type, differ substantially in their allele frequencies for the growth-associated NCAPG p.Ile442Met locus.

Evaluation of a replacement method for mammary gland biopsies by comparing gene expression in udder tissue and mammary epithelial cells isolated from milk.

Somatic cells isolated from milk offer an attractive non-invasive replacement of invasive udder biopsies for monitoring bovine mammary gland metabolism. However, for metabolic gene expression studies the mammary gland epithelial cells (MEC) isolated from milk have to be purified from the non-epithelial leukocyte fraction in milk samples. In our study, enrichment of MEC by using anti-cytokeratin peptide 18 (KRT18) antibody coated magnetic beads was evaluated. MEC showed a substantially increased expression of the epithelial-cell-specific KRT18 gene compared to udder tissue. The expression levels of genes specific for mammary gland epithelial cells (CSN3 and LALBA) showed a significant positive correlation in MEC and also in udder tissue. However, no significant correlation of the expression of a specific gene was found between udder and MEC samples. Therefore, MEC isolated from total milk samples via KRT18 antibodies probably do not reflect the true metabolic situation of the bovine udder. Thus, quantitative gene expression profiling of MEC isolated via KRT18 antibodies has to be interpreted carefully with respect to the situation in the udder.

The SNP c.1326T>G in the non-SMC condensin I complex, subunit G (NCAPG) gene encoding a p.Ile442Met variant is associated with an increase in body frame size at puberty in cattle.

Recently, we had located a bovine carcass weight QTL, CW-2, to a 591-kb interval on BTA6 and have identified the SNP c.1326T>G in the NCAPG (non-SMC condensin I complex, subunit G) gene that leads to the amino acid change p.Ile442Met in the NCAPG protein, which is a candidate causative variation. Here, we examined the association of the NCAPG:c.1326T>G locus with linear skeletal measurements of growth-associated traits during adolescence, which is a period of intensive growth, using two historically and geographically distant cattle populations: 792 Japanese Black steers and 161 F(2) bulls of an experimental cross from Charolais and German Holstein. In both populations, the SNP NCAPG:c.1326T>G was associated with each component of body frame size: height, length and width at puberty. The associations of CW-2 with height- and length-associated traits were observed at an earlier growth period compared to the associations with thickness- and width-associated traits, indicating that the primary effect of the CW-2 QTL may possibly be exerted on skeletal growth. The significant associations of the NCAPG:c.1326T>G locus with growth-associated skeletal measurements are similar to the effects of the syntenic region on human chromosome 4 that are associated with adult height in humans, supporting the hypothesis that CW-2 is analogous to the human locus and pointing to a conserved growth-associated locus or chromosomal region present in both species.

A genetic predisposition for bovine neonatal pancytopenia is not due to mutations in coagulation factor XI.

Bovine neonatal pancytopenia (BNP) is a newly emerging disease in many European countries that causes haemorrhagic diathesis and mortality in neonatal calves. This study tested the hypothesis that genetic factors might be involved in BNP, since genetic defects resulting in coagulation disorders have been described in many species, including cattle. A familial pattern of occurrence of BNP cases was observed in an experimental population of cattle in Germany and BNP was diagnosed in nine calves on an experimental dairy herd from May 2007 to December 2009. All affected calves were descendents of a single F(1) sire in a specific F(2) resource population generated from Charolais and German Holstein bloodlines. Sequence analysis of the bovine coagulation factor XI (F11) gene as a functional candidate gene for BNP revealed an unusually high number of non-synonymous mutations within the gene compared to a whole genome mutation screen in cattle targetting random sequences. However, none of the mutations in the F11 gene were concordant with BNP status. Although these data and further pedigree analysis excluded a simple mode of inheritance of the BNP phenotype, there was a statistically significant (P=0.0001) accumulation of BNP cases in the specific pedigree examined, suggesting that a genetic component is involved in the development of BNP.

Differences in milk production, glucose metabolism, and carcass composition of 2 Charolais x Holstein F2 families derived from reciprocal paternal and maternal grandsire crosses.

Two F(2) Charolais x German Holstein families comprising full and half sibs share identical but reciprocal paternal and maternal Charolais grandfathers differ in milk production. We hypothesized that differences in milk production were related to differences in nutritional partitioning revealed by glucose metabolism and carcass composition. In 18F(2) cows originating from mating Charolais bulls to German Holstein cows and a following intercross of the F(1) individuals (n=9 each for family Ab and Ba; capital letters indicate the paternal and lowercase letter the maternal grandsire), glucose tolerance tests were performed at 10 d before calving and 30 and 93 d in milk (DIM) during second lactation. Glucose half-time as well as areas under the concentration curve for plasma glucose and insulin were calculated. At 94 DIM cows were infused intravenously with 18.3 micromol of d-[U-(13)C(6)]glucose/kg(0.75) of BW, and blood samples were taken to measure rate of glucose appearance and glucose oxidation as well as plasma concentrations of metabolites and hormones. Cows were slaughtered at 100 DIM and carcass size and composition was evaluated. Liver samples were taken to measure glycogen and fat content, gene expression levels, and enzyme activities of pyruvate carboxylase, phosphoenolpyruvate carboxykinase, and glucose 6-phosphatase as well as gene expression of glucose transporter 2. Milk yield was higher and milk protein content at 30 DIM was lower in Ba than in Ab cows. Glucose half-life was higher but insulin secretion after glucose challenge was lower in Ba than in Ab cows. Cows of Ab showed higher glucose oxidation, and plasma concentrations at 94 DIM were lower for glucose and insulin, whereas beta-hydroxybutyrate was higher in Ba cows. Hepatic gene expression of pyruvate carboxylase, glucose 6-phosphatase, and glucose transporter 2 were higher whereas phosphoenolpyruvate carboxykinase activities were lower in Ba than in Ab cows. Carcass weight as well as fat content of the carcass were higher in Ab than in Ba cows, whereas mammary gland mass was lower in Ab than in Ba cows. Fat classification indicated leaner carcass composition in Ba than in Ab cows. In conclusion, the 2 families showed remarkable differences in milk production that were accompanied by changes in glucose metabolism and body composition, indicating capacity for milk production as main metabolic driving force. Sex chromosomal effects provide an important regulatory mechanism for milk performance and nutrient partitioning that requires further investigation.

Evolutionary break point analysis between the proximal half of bovine chromosome 27 and conserved segments of the human genome.

The proximal half of Bos taurus chromosome 27 (BTA27prox) delimited by microsatellite markers BM3507 and CSSM043 reveals complex rearrangements compared to its corresponding Homo sapiens chromosome (HSA) fragments. A comparative mapping approach combining somatic and radiation hybrid cell mapping techniques and related cytogenetic data resulted in an improved physical map for BTA27prox, which provides candidate genes for several important economic traits. The generated comprehensive map includes anchor loci for 103 genes and microsatellite markers. Mapping of genes proximal to BM3507 matching a region from 0.60 to 2.78 megabase pairs (Mb) of HSA8 confirmed recent sequence annotations on BTA27. Assignments of loci predicted to be on BTA27 to BTA1, BTA8, and BTA17 narrowed down evolutionary chromosome break points compared with corresponding chromosome segments in human. New physical anchors obtained in this study confirm in more detail the described evolutionary conservation between the proximal half of BTA27 and homologous segments of HSA4 and HSA8 and will contribute to the completion of the cattle DNA genome sequence.

An investigation into the genetic background of coat colour dilution in a Charolais x German Holstein F2 resource population.

The molecular background of many loci affecting coat colour inheritance in cattle is still incompletely characterized, although it is known that coat colour results from the joint effects of several loci, e.g. agouti, extension and dilution. Dilution alleles are responsible for a dilution effect on the original coat colour of an individual, which is determined by the agouti and extension loci. Different loci affecting dilution of pigment are suggested in Charolais (Dc) and Simmental (Ds). To enable chromosomal mapping of the Dc mutation, 133 animals from an F2 full-sib resource population generated from a cross of Charolais and German Holstein were scored for the coat colour dilution phenotype. Linkage analysis covering all autosomes revealed a significant linkage of the dilution phenotype with microsatellite markers on bovine chromosome 5. No recombination was observed between marker ETH10 and the Dc locus. Positional and functional information identified the bovine silver homolog (SILV) gene as a candidate for the Dc mutation. Results from comparative sequencing of the SILV gene in individuals with different dilution coat colour phenotypes confirmed the presence of a c.64G>A non-synonymous mutation, which had previously been identified in the Charolais breed. The alleles at this locus were associated with coat colour dilution in this study. However, further investigation of colour inheritance within the F2 resource population indicated that a single diallelic mutation in the SILV gene cannot explain the total observed variation of coat colour dilution.

Forward to a detailed sequence map of bovine chromosome 6.

Numerous QTL for a variety of phenotypic traits in dairy and beef cattle have been mapped on bovine chromosome 6 (BTA6). The complete and validated information on the molecular genome organization is an essential prerequisite for the conclusive identification of the causative sequence variation underlying the QTL. In our study we describe efforts to improve the genomic sequence map assembly of BTA6 by filling-in gaps and by suggesting sequence contig rearrangements. This is achieved by the generation and in silico mapping of BAC-end sequences (BESs) from clones containing sequences placed on our high-resolution radiation hybrid (RH) map of BTA6 onto the genome sequence map. Linking high-resolution RH mapping with in silico mapping of BESs on BTA6 enabled the detection of discrepancies in chromosomal assignments of genome sequence contigs and improved the resolution of non-conclusive assignments on the genome sequence assembly. Furthermore, 37% of BESs enabled chromosomal assignment of contigs previously unassigned. Anchoring of 66% of BESs onto HSA4 confirmed the synteny of the respective region of BTA6 including the known evolutionary breakpoints. The BESs will play an important role in the ongoing efforts to complete the sequence of the bovine genome and will also provide a source for the identification of new polymorphic sites in the genome sequence to resolve QTL-containing intervals.

A radiation hybrid map of river buffalo (Bubalus bubalis) chromosome 7 and comparative mapping to the cattle and human genomes.

A preliminary radiation hybrid (RH) map containing 50 loci on chromosome 7 of the domestic river buffalo Bubalus bubalis (BBU; 2n = 50) was constructed based on a comparative mapping approach. The RH map of BBU7 includes thirty-seven gene markers and thirteen microsatellites. All loci have been previously assigned to Bos taurus (BTA) chromosome BTA6, which is known for its association with several economically important milk production traits in cattle. The map consists of two linkage groups spanning a total length of 627.9 cR(5,000). Comparative analysis of the BBU7 RH(5,000) map with BTA6 in cattle gave new evidence for strong similarity between the two chromosomes over their entire length and exposed minor differences in locus order. Comparison of the BBU7 RH(5,000) map with the Homo sapiens (HSA) genome revealed similarity with a large chromosome segment of HSA4. Comparative analysis of loci in both species revealed more variability than previously known in gene order and several chromosome rearrangements including centromere relocation. The data obtained in our study define the evolutionarily conserved segment on BBU7 and HSA4 to be between 3.5 megabases (Mb) and 115.8 Mb in the HSA4 (genome build 36) DNA sequence.

Search for pleiotropic QTL on chromosome BTA6 affecting yield traits of milk production.

The primary aim of this study was to investigate whether previous findings of similar quantitative trait loci (QTL) positions for correlated yield traits are due to a pleiotropic QTL. We applied a multitrait variance component based QTL mapping method to a dataset involving five granddaughter families from the German Holstein dairy cattle population. The marker map contained 16 microsatellite markers, distributed across chromosome BTA6. A chromosomewise significance threshold was used, because BTA6 is known to harbor QTL for several milk traits. To evaluate the results from the multivariate, across-family analysis, we also conducted single-family analyses using the least squares method of QTL estimation. The results provided two significant QTL findings at 49 and 64 cM for milk yield in different families and putative QTL at 68 cM for fat yield and at 71 cM for protein yield in another family. The results for fat and protein yield were confirmed by a univariate, across-family variance components analysis. The multivariate analysis of three bivariate trait combinations resulted in a significant pleiotropic QTL finding at 68 cM for fat yield and protein yield, bracketed by markers TGLA37 and FBN13. The estimates of variance contribution due to this QTL were 23% and 25%, respectively.