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Improving feed efficiency (FE) is a major goal for the livestock industry. Previously, we have identified 48 SNP markers distributed over 32 genes significantly associated with residual feed intake (RFI) in Israeli Holstein male calves, the most significant of which are located in the bovine FABP4 gene. In the present study, we tested associations of eight of the FABP4 markers with RFI and feed conversion ratio (FCR), along with milk composition and feeding behavioral traits, in 114 lactating Israeli Holstein cows. Large allele effects were found, along with large contributions of FABP4 markers to the phenotypic variation [mean contribution of all significant markers ( P < 0.05), 15.4 and 12.0% for RFI and FCR, respectively] and genotypic variation [means of all significant markers ( P < 0.05), 75.7 and 32.4% in RFI and FCR, respectively]. However, the association of all significant FABP4 markers with FE and milk content traits was found in opposite directions, such that improved FE was accompanied by decreased milk content. Hence, before inclusion in breeding programs, the gain in FE must be economically balanced with the loss in milk contents. On the other hand, these findings imply that in any current improvement program concentrated on milk traits alone, without taking into account the effect on FE, the progress in milk composition is probably accompanied by deterioration of FE. These results, if confirmed in other populations and breeds, set FABP4 as a prime candidate in any marker-assisted selection program targeting FE as a whole and RFI in particular.

We report on a complete genome scan for quantitative trait loci (QTL) affecting milk protein percentage (PP) in the Italian Holstein-Friesian cattle population, applying a selective DNA pooling strategy in a daughter design. Ten Holstein-Friesian sires were chosen, and for each sire, about 200 daughters, each from the high and low tails of estimated breeding value for PP, were used to construct milk DNA pools. Sires and pools were genotyped for 181 dinucleotide microsatellites covering all cattle autosomes. Sire marker allele frequencies in the pools were obtained by shadow correction of peak height in the electropherograms. After quality control, pool data from eight sires were used for all subsequent analyses. The QTL heterozygosity estimate was lower than that of similar studies in other cattle populations. Multiple marker mapping identified 19 QTL located on 14 chromosomes (BTA1, 2, 3, 4, 5, 6, 8, 9, 12, 14, 17, 20, 23 and 27). The sires were also genotyped for seven polymorphic sites in six candidate genes (ABCG2, SPP1, casein kappa, DGAT1, GHR and PRLR) located within QTL regions of BTA6, 14 and 20 found in this study. The results confirmed or excluded the involvement of some of the analysed markers as the causative polymorphic sites of the identified QTL. The QTL identified, combined with genotype data of these candidate genes, will help to identify other quantitative trait genes and clarify the complex QTL patterns observed for a few chromosomes. Overall, the results are consistent with the Italian Holstein population having been under long-term selection for high PP., (© 2012 The Authors, Animal Genetics © 2012 Stichting International Foundation for Animal Genetics.)

Mastitis is an important and common dairy cattle disease affecting milk yield, quality, and consumer safety as well as cheese yields and quality. Animal welfare and residues of the antibiotics used to treat mastitis cause public concern. Considerable genetic variation may allow selection for increased resistance to mastitis. Because of high genetic correlation to milk somatic cell score (SCS), SCS can serve as a surrogate trait for mastitis resistance. The present study intended to identify quantitative trait loci (QTL) affecting SCS in Israeli and Italian Holstein dairy cattle (IsH and ItH, respectively), using selective DNA pooling with single and multiple marker mapping. Milk samples of 4,788 daughters of 6 IsH and 7 ItH sires were used to construct sire-family high- and low-tail pools, which were genotyped at 123 (IsH) and 133 (ItH) microsatellite markers. Shadow correction was used to obtain pool allele frequency estimates. Frequency difference between the tails and empirical standard error of D, SE(D), were used to obtain P-values. All markers significant by single marker mapping were also significant by multiple marker mapping, but not vice versa. Combining both populations, 22 QTL on 21 chromosomes were identified; all corresponded to previous reports in the literature. Confidence intervals were set by chi-squared drop method. Heterozygosity of QTL was estimated at 44.2%. Allele substitution effects ranged from 1,782 to 4,930 cells/mL in estimated breeding value somatic cell count units. Most (80%) of the observed variation in estimated breeding value somatic cell score could be explained by the QTL identified under the stringent criteria. The results found here can be used as a basis for further genome-wide association studies for the same trait., (Copyright © 2010 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

To fine map a quantitative trait locus (QTL) affecting milk production traits previously associated with microsatellite RM188, we implemented an interval mapping analysis by using microsatellite markers in a large Israeli Holstein half-sib sire family, and linkage disequilibrium (LD) mapping in a large set of US Holstein bulls. Interval mapping located the target QTL to the near vicinity of RM188. For the LD mapping, we identified 42 single nucleotide polymorphisms (SNP) in 15 genes in a 12-Mb region on bovine chromosome 4. A total of 24 tag SNP were genotyped in 882 bulls belonging to the University of California Davis archival collection of Holstein bull DNA samples with predicted transmitted ability phenotypes. Marker-to-marker LD analysis revealed 2 LD blocks, with intrablock r(2) values of 0.10 and 0.46, respectively; outside the blocks, r(2) values ranged from 0.002 to 0.23. A standard additive/dominance model using the generalized linear model procedure of SAS and the regression module of HelixTree software were used to test marker-trait associations. Single nucleotide polymorphism 9 on ARL4A, SNP10 on XR&#95;027435.1, SNP12 on ETV1, SNP21 on SNX13, and SNP24 were significantly associated with milk production traits. We propose the interval encompassing ARL4A and SNX13 genes as a candidate region in bovine chromosome 4 for a concordant QTL related to milk protein traits in dairy cattle. Functional studies are needed to confirm this result.

Protein yield (PY) is currently the major economic product of the dairy herd. Genome-wide scans for quantitative trait loci (QTL) affecting milk yield (MY) and milk protein percentage (PP) suggest that of the loci affecting the 2 traits, about 1/4 exclusively affect MY, 1/4 exclusively affect PP, and half affect both traits. Because PY is the product of MY and PP, it is of interest to evaluate the expected effects on PY of marker-assisted selection (MAS) applied to these 3 classes of QTL. It is clear that selection for the appropriate allele at QTL exclusively affecting MY or PP will have a positive effect on PY. The question arises as to the effect of MAS directed at QTL affecting both MY and PP. Because the observed genetic correlation of about -0.5 between MY and PP must be generated by these loci, and because they comprise about half the total number of loci affecting the 2 traits, it can be inferred that the genetic correlation between MY and PP at loci affecting both traits is close to -1.0. This seems to imply that generally such loci would be neutral in their effects on PY. In the present study, biometrical expressions originally developed to describe the relationships of MY, fat percentage, and fat yield were adapted to describe the relationships of MY, PP, and PY. The resultant expressions were validated by showing that they correctly predicted the observed phenotypic standard deviation and heritability of PY, and the vastly different genetic correlations of PY with MY (very high positive) and of PY with PP (very low positive). Contrary to initial impressions, further biometrical analysis of the projected effects on PY of MAS at the loci affecting both traits, showed that even under the assumption that the genetic correlation between MY and PP at these loci is -1.0, selection for the allele favoring MY will have a strong positive effect on PY, whereas selection for the allele favoring PP will have an equal but opposite negative effect on PY. These diametrically opposed effects are due to the lower genetic coefficient variation of PP compared with MY. It is speculated that the reduced coefficient of variation of PP may be because of more stringent homeostatic buffering of milk composition compared with milk yield.

Fibroblast growth factor 2 (FGF2) is expressed in the bovine mammary gland and may play a role in the development and reorganization of the mammary gland. It is also expressed by the uterine endometrium throughout the estrous cycle and early pregnancy. The FGF2 was chosen for this study because it regulates the expression of interferon-tau, a key member of the signal transduction pathway involved in milk production. In previous studies, we reported the association of several genes in this pathway with milk production and health traits in dairy cattle. The objective of this study was to examine the association of FGF2 polymorphisms with milk composition, somatic cell score, and productive life in 3 Holstein cattle populations from the United States and Israel. The pooled DNA sequencing approach was used to identify single nucleotide polymorphisms (SNP) in FGF2. Sequencing of a total of 6.4 kb including 3 exons of the gene revealed only one SNP (A/G) in intron 1 at position 11646. This SNP was investigated for association with production traits in 2,773 individuals from 3 Holstein populations: the granddaughter-design Cooperative Dairy DNA Repository and the daughter-design University of Wisconsin populations from the United States and a daughter-design population from Israel. For both the Israeli and the UW populations, FGF2 variants were associated with fat yield and percentage, somatic cell score, and productive life with significant dominance and complete dominance effects. For the Cooperative Dairy DNA Repository population, no significant associations were observed for the examined traits. Given that FGF2 was chosen for this study because of its role in the interferon-tau signal transduction pathway and was found to be associated with production traits, results suggest that the candidate pathway could be an attractive strategy to search for candidate quantitative trait genes.

Although numerous quantitative trait loci (QTL) mapping studies involving milk protein percent (PP), milk yield (MY), and protein yield (PY) have been carried out, there has not been any systematic evaluation of the effects of individual QTL on these 3 interrelated traits. Consequently, the aim of the present study was to investigate the effects on MY and PY of QTL for PP previously mapped in various laboratories. The study, based on selective DNA pooling of milk samples, included 10 Israeli Holstein artificial insemination bulls, each the sire of 1,800 or more milk-recorded daughters. For each sire-trait combination across the 10 sires, milk samples of the highest and lowest daughters with respect to estimated breeding values for PP, PY, and MY were collected for pooling. A total of 134 dinucleotide microsatellites distributed over 25 bovine autosomes were used. An empirical standard error for marker-QTL linkage testing was calculated based on the variation among split samples within the same tail. Threshold comparison-wise error rate P-values were set to control proportion of false positives at P = 0.10 level for declaring significant effects at the marker-trait level. Estimates of the number of true null hypotheses for each trait were obtained from the histogram of marker comparison-wise error rate P-values. Based on these estimates, effective power of the experiment at the marker-trait level was estimated as 0.75, 0.41, and 0.73 for PP, PY, and MY. The proportion of heterozygosity at the QTL was estimated as 0.46, 0.39, and 0.40, respectively. After correcting for incomplete power and proportion of false positives, it was estimated that 38.7 and 37.5% of the markers affecting PP and MY, respectively, also affected PY. Of the markers affecting PY, 68.9 and 76.5%, respectively, also affected PP and MY. Apparently, none of the significant markers affected PY exclusively, and only 6.5 and 16.0%, respectively, affected PP or MY exclusively. Thus, almost all significant markers, and by inference almost all QTL, had effects on at least 2 of the 3 traits.

Quantitative trait loci (QTL) mapping projects have been implemented mainly in the Holstein dairy cattle breed for several traits. The aim of this study is to map QTL for milk yield (MY) and milk protein percent (PP) in the Brown Swiss cattle populations of Austria, Germany, and Italy, considered in this study as a single population. A selective DNA pooling approach using milk samples was applied to map QTL in 10 paternal half-sib daughter families with offspring spanning from 1,000 to 3,600 individuals per family. Three families were sampled in Germany, 3 in Italy, 1 in Austria and 3 jointly in Austria and Italy. The pools comprised the 200 highest and 200 lowest performing daughters, ranked by dam-corrected estimated breeding value for each sire-trait combination. For each tail, 2 independent pools, each of 100 randomly chosen daughters, were constructed. Sire marker allele frequencies were obtained by densitometry and shadow correction analyses of 172 genome-wide allocated autosomal markers. Particular emphasis was placed on Bos taurus chromosomes 3, 6, 14, and 20. Marker association for MY and PP with a 10% false discovery rate resulted in nominal P-values of 0.071 and 0.073 for MY and PP, respectively. Sire marker association tested at a 20% false discovery rate (within significant markers) yielded nominal P-values of 0.031 and 0.036 for MY and PP, respectively. There were a total of 36 significant markers for MY, 33 for PP, and 24 for both traits; 75 markers were not significant for any of the traits. Of the 43 QTL regions found in the present study, 10 affected PP only, 8 affected MY only, and 25 affected MY and PP. Remarkably, all 8 QTL regions that affected only MY in the Brown Swiss, also affected MY in research reported in 3 Web-based QTL maps used for comparison with the findings of this study (http://www.vetsci.usyd.edu.au/reprogen/QTL&#95;Map/; http://www.animalgenome.org/QTLdb/cattle.html; http://bovineqtl.tamu.edu/). Similarly, all 10 QTL regions in the Brown Swiss that affected PP only, affected only PP in the databases. Thus, many QTL appear to be common to Brown Swiss and other breeds in the databases (mainly Holstein), and an appreciable fraction of QTL appears to affect MY or PP primarily or exclusively, with little or no effect on the other trait. Although QTL information available today in the Brown Swiss population can be utilized only in a within family marker-assisted selection approach, knowledge of QTL segregating in the whole population should boost gene identification and ultimately the implementation and efficiency of an individual genomic program.

Bovine mastitis caused by Streptococcus agalactiae is mainly subclinical and therefore can be diagnosed only in the laboratory. We developed a polymerase chain reaction (PCR)-based method for specific and sensitive detection of S. agalactiae in raw milk. The specificity of the PCR reaction is based on unique S. agalactiae DNA sequences within the 16S subunit of the rRNA genes. Two pairs of sequences were used as positive controls; general streptococci primers, which anneal to conserved areas within the 16S rRNA subunit gene, and primers, which anneal to sequences within bovine mitochondrial DNA. The method of detection includes selective enrichment of S. agalactiae in the milk sample, followed by DNA extraction using a rapid and simple procedure developed for this purpose, and specific PCR reaction with appropriate controls. The method enables the detection of one bacterium in 1 ml of raw milk. The method developed can be easily incorporated as part of routine screening of bulk milk collection tanks for early detection of infected cows in a herd.

"Selective DNA pooling" accomplishes quantitative trait locus (QTL) mapping through densitometric estimates of marker allele frequencies in pooled DNA samples of phenotypically extreme individuals. With poly(TG) microsatellites, such estimates are confounded by "shadow" ("stutter") bands. A correction procedure was developed on the basis of an observed linear regression between shadow band intensity and allele TG repeat number. Using this procedure, a selective DNA pooling study with respect to milk protein percentage was implemented in Israel-Holstein dairy cattle. Pools were prepared from milk samples of high and low daughters of each of seven sires and genotyped with respect to 11 markers. Highly significant associations with milk protein percentage were found for 5 of the markers; 4 of these markers confirmed previous reports. Selective DNA pooling accessed 80.6 and 48.3%, respectively, of the information that would have been available through individual selective genotyping or total population genotyping. In effect, the statistical power of 45,600 individual genotypings was obtained from 328 pool genotypings. This methodology can make genome-wide mapping of QTL accessible to moderately sized breeding organizations.

Somatic cells in milk were used as a source of DNA and as a substrate for the polymerase chain reaction. Successful DNA extraction required a minimum total of 17 x 10(6) cells in the milk sample. The DNA yield per cell was highly variable but comparable, on average, with the yield from peripheral blood leukocytes. In all cases, direct polymerase chain reaction on milk samples, using a variety of primer pairs, yielded amplified products of correct size that were identical to those obtained by polymerase chain reaction of purified DNA extracted from milk or blood. Milk and DNA extracted from milk served as substrates for direct sequencing of part of the bovine growth hormone gene. Milk samples with added preservative were stored over 200 d without effect on the polymerase chain reaction. Thus, milk can often substitute for blood as a source of DNA for Southern blot analysis and is a preferred substrate for the polymerase chain reaction. The technical convenience of milk as a source of DNA can be expected to increase the field of application of marker-based methods for genetic analysis and genetic improvement of economic traits in dairy cattle.