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Whole blood stimulation assay (WBA) has been widely used to study production of the pro-inflammatory cytokine, tumor necrosis factor-alpha (TNF-alpha) and is considered a relatively good predictor for the in vivo release of TNF-a during endotoximia in dairy cattle. However, it requires a standardized and optimized dose of lipopolysaccharides (LPS) to simulate the in vivo cytokine response during WBA when using killed bacteria. Moreover, if an association between genome (e. g., Quantitative trait loci) and ex vivo TNF-alpha responsiveness can be established; it helps to reduce mastitis in dairy cattle using selective breeding. Three experiment scenarios for pathogen dose, stimulation time and estimation of genetic variables effect in ex vivo TNF-alpha production were carried out to investigate differences of TNF-alpha responsiveness in early lactation in 4, 38 and 40 Danish Holstein Friesian dairy cows respectively. Statistical analysis done using ANOVA, t.test and lm functions in R software. Results showed that, ex vivo TNF-alpha response to E. coli is dose and time dependent and a dose of 2.5 x 10(6) CFU/mL for 3.5 h is optimal for observing TNF-alpha response after ex vivo stimulation.Relatively consistent inter-individual cow differences in the TNF-alpha response ex vivo were observed from week 3 to week 8. No statistically significant association were found between the ex vivo TNF-alpha responsiveness and the two E. call associated mastitis QTLs which may be due to the low number of animals (n =) in the study.

Several quantitative trait loci (QTL) affecting mastitis incidence and mastitis-related traits such as somatic cell score exist in dairy cows. Previously, QTL haplotypes associated with susceptibility to Escherichia coli mastitis in Nordic Holstein-Friesian (HF) cows were identified on Bos taurus autosome 9. In the present study, we induced experimental E. coli mastitis in Danish HF cows to investigate the effect of 2 E. coli mastitis-associated QTL haplotypes on the cows' disease phenotypes and recovery in early lactation. Thirty-two cows were divided in 2 groups bearing haplotypes with either low (HL) or high (HH) susceptibility to E. coli . In addition, biopsies (liver and udder) were collected from half of the cows (n=16), resulting in a 2 × 2 factorial design, with haplotype being one factor (HL vs. HH) and biopsy being the other factor (biopsies vs. no biopsies). Each cow was inoculated with a low E. coli dose (20 to 40 cfu) in one front quarter at time 0h. Liver biopsies were collected at −144, 12, 24, and 192h; udder biopsies were collected at 24h and 192h post- E. coli inoculation. The clinical parameters: feed intake, milk yield, body temperature, heart rate, respiration rate, rumen motility; and the paraclinical parameters: bacterial counts, somatic cell count (SCC), and milk amyloid A levels in milk; and white blood cell count, polymorphonuclear neutrophilic leukocyte (PMNL) count, and serum amyloid A levels in blood were recorded at different time points post- E. coli inoculation. Escherichia coli inoculation changed the clinical and paraclinical parameters in all cows except one that was not infected. Clinically, the HH group tended to have higher body temperature and heart rate than the HL group did. Paraclinically, the HL group had faster PMNL recruitment and SCC recovery than the HH group did. However, we also found interactions between the effects of haplotype and biopsy for body temperature, heart rate, and PMNL. In conclusion, when challenged with E. coli mastitis, HF cows with the specific Bos taurus autosome 9-located QTL haplotypes were associated with differences in leukocyte kinetics, with low-susceptibility cows having faster blood PMNL recruitment and SCC recovery and a tendency for a milder clinical response than the high-susceptibility cows did.

Following the recent years' increase in herd size, the awareness of a group of cows with a generally lowered health and production level, the “loser cows,” has arisen in Denmark. The aim of this study was to estimate genetic correlations between the loser cow score, 305d protein yield and realized productive life (RPL) and inbreeding depression for the loser cow score. Estimation of genetic correlations and inbreeding depression was performed on 2644 first-lactation cows and 4914 multiple-lactation cows, respectively. We found no significant genetic correlation between the loser cow score and the inbreeding level or between the loser cow score and protein yield. However, the loser cow score was favorably correlated with RPL (−0.53). This implies that the prevalence of loser cows is neither a result of inbreeding depression nor an undesirable side effect of selecting for increased production.

We establish a TaqMan-based assay panel for genotyping single-nucleotide polymorphisms in rainbow trout and steelhead (Oncorhynchus mykiss). We develop 22 novel single-nucleotide polymorphism markers based on new steelhead sequence data and on assays from sister taxa. Additionally, we adapt 154 previously developed markers to the TaqMan platform. At the beginning of this study, 59 SNPs with TaqMan assays were available to the scientific community. By adding 176 additional TaqMan assays to this number, we greatly expand the biological applications of TaqMan genotyping within both population genetics and quantitative genetics.

Over the last few years, an increasing awareness has arisen in Denmark of the existence of cows with a generally lowered health and production status, referred to as "loser cows." A previous study has estimated that the overall prevalence of loser cows in Danish Holstein herds is 3.2%. The aim of this study was to estimate genetic and phenotypic parameters for the loser cow state and the underlying traits: lameness, hock lesions, other cutaneous lesions, and condition of hair coat. Records on 6,098 cows were analyzed with an animal model including fixed effects of herd, season of scoring and location of scoring, age at first calving, lactation stage, and parity in addition to additive genetic effects and permanent environmental effects. The heritability of the loser cow score was 0.08 and for the underlying traits the heritability ranged from 0.05 to 0.12. The genetic correlations between various pairs of traits included in the loser cow score ranged from 0.04 to 0.68 and the phenotypic correlations ranged from 0.09 to 0.21. The genetic and phenotypic correlations between the loser cow score and the underlying traits ranged from 0.25 to 0.89 and 0.20 to 0.85, respectively, supporting the concept of the loser cow score. The traits included in the loser cow score are easy to assess and all showed genetic variation. They are therefore suitable for inclusion in a total merit index aimed at breeding for more robust cows.

Key words: baleen whale, kinship, Mysteceti, STR lociInconservationgenetics,itisbecomingincreasinglyclear that parameters derived over evolutionarytime scales may not apply to much shorter ecolog-ical timescales (Palsboll 1999). Molecular geneticmethods can be employed to estimate parametersonanecological timescale ifthe focusis aimedonlyat recently diverged lineages, i.e., among individu-als as opposed to among populations. Theseextensions of current approaches are vital to alignthe application of molecular genetics to contem-porary issues in conservation. In order to estimatethe degree of kinship in a reliable manner, an ade-quate number of loci must be analyzed per indi-vidual. Ensuring an adequate number of locidecreases/eliminates the interference of other levelsof relatives, and compensates for the exponentialincrease in the number of pair-wise comparisonswhen the sample size increases. Towards this end,we presented, in this note, an additional 17 poly-morphic microsatellite loci, which, originated froma di-, tri- and tetra-nucleotide microsatellite locienriched library constructed from humpbackwhale, Megaptera novaeangliae, and fin whale,Balaenoptera physalus, genomic DNA, using theprotocol previously described in Palsboll et al.(1997) and Be´rube´et al. (2000).More specifically, the microsatellite loci wereisolated from size-selected total-cell DNAextracted from humpback whale and fin whaleusing the approach described by Rassmann andcolleagues (1991). Recombinant colonies (dotblotted to a ZetaProbe

Numerically small but statistically significant genetic differentiation has been found in many marine fish species despite very large census population sizes and absence of obvious barriers to migrating individuals. Analyses of morphological traits have previously identified local spawning groups of herring (Clupea harengus L.) in the environmentally heterogeneous Baltic Sea, whereas allozyme markers have not revealed differentiation. We analysed variation at nine microsatellite loci in 24 samples of spring-spawning herring collected at 11 spawning locations throughout the Baltic Sea. Significant temporal differentiation was observed at two locations, which we ascribe to sympatrically spawning but genetically divergent 'spawning waves'. Significant differentiation was also present on a geographical scale, though pairwise F(ST) values were generally low, not exceeding 0.027. Partial Mantel tests showed no isolation by geographical distance, but significant associations were observed between genetic differentiation and environmental parameters (salinity and surface temperature) (0.001 < P < or = 0.099), though these outcomes were driven mainly by populations in the southwestern Baltic Sea, which also exhibits the steepest environmental gradients. Application of a novel method for detecting barriers to gene flow by combining geographical coordinates and genetic differentiation allowed us to identify two zones of lowered gene flow. These zones were concordant with the separation of the Baltic Sea into major basins, with environmental gradients and with differences in migration behaviour. We suggest that similar use of landscape genetics approaches may increase the understanding of the biological significance of genetic differentiation in other marine fishes.

Temporal sampling within the spring-spawning season has revealed differentiation in length-at-age in herring at Rügen and differentiation in, e.g., Anisakis infestation rate, otolith microstructure, and gillraker counts in Gdańsk Bay, leading to the expectation that spawning waves consist of distinct herring populations. We tested this expectation by analysing genetic variation at nine microsatellite loci in samples collected at different times during the March to May spawning season in 2 consecutive years, 2002 and 2003. Length-at-age, mean length, and age distributions were compared among samples within locations but did not show consistent temporal patterns. Pairwise genetic differentiation among temporal samples within season was low and non-significant in the Gdańsk Bay (0 < FST < 0.0025) but higher among Rügen samples (0.0008 < FST < 0.0113). Samples from Rügen collected in 2002 differed significantly from each other, and individual assignment tests showed increased divergence with time. Differentiation was not confounded by effects of age class or sex. We conclude that spawning waves are not genetically differentiated among Gdańsk samples based on factors analysed in this study, whereas genetically distinct but sympatric spawning populations may be found at Rügen.

The mammalian liver works to keep the body in a state of homeostasis and plays an important role in systemic acute phase response to infections. In this study we investigated the bovine hepatic acute phase response at the gene transcription level in dairy cows with experimentally Escherichia coli -induced mastitis. At time = 0, each of 16 periparturient dairy cows received 20–40 colony-forming units of live E. coli in one front quarter of the udder. A time series of liver biopsies was collected at −144, 12, 24, and 192 h relative to time of inoculation. Changes in transcription levels in response to E. coli inoculation were analyzed using the Bovine Genome Array and tested significant for 408 transcripts over the time series [adjusted p ≤0.05, abs(fold-change)>2]. After 2-D clustering, transcripts represented three distinct transcription profiles: 1) regulation of gene transcription and apoptosis, 2) responses to cellular stress invoked by reactive metabolites, and 3) metabolism and turnover of proteins. The results showed that the liver went through a period of perturbations to its normal homeostatic condition during the first 24 h following the E. coli -induced intra-mammary inflammation. In previous studies, bacterial lipopolysaccharide, LPS, was used for intramammary stimulation to mimic E. coli infection. Comparing responses to LPS and E. coli , induced biochemical processes were similar but not identical (94 and 85% similarity between corresponding samples at early and late acute phase, respectively), but their kinetics were not. A notable difference concerned transcription of factors associated with oxidative stress in E. coli -induced liver responses.

The spatial structuring of intraspecific genetic diversity is the result of random genetic drift, natural selection, migration, mutation, and their interaction with historical processes. The contribution of each has been typically difficult to estimate, but recent advances in statistical genetics have provided valuable new investigative tools to tackle such complexity. Using a combination of such methods, we examined the roles of environment (i.e., natural selection), random genetic processes (i.e., drift), and demography and life histories (e.g., feeding migrations) on population structure of a widely distributed and abundant marine pelagic fish of economic importance, Atlantic herring (Clupea harengus). Individuals were collected during peak spawning time from 19 spawning locations spanning the region from the western North Sea to the eastern Baltic Sea (N= 1859, eight microsatellite loci). We carried out separate analyses of neutral and selected genetic variation, which allowed us to establish that the two most important factors affecting population structure were selection due to salinity at spawning sites and feeding migrations. The genetic signal left by the demographic history of herring, on the other hand, seems to have been largely eroded, which is not surprising given the large reproductive potential and presumed enormous local effective population sizes of pelagic fish that constrain the effect of stochastic processes. The approach we used can in principle be applied to any abundant and widely distributed aquatic or terrestrial species The spatial structuring of intraspecific genetic diversity is the result of random genetic drift, natural selection, migration, mutation, and their interaction with historical processes. The contribution of each has been typically difficult to estimate, but recent advances in statistical genetics have provided valuable new investigative tools to tackle such complexity. Using a combination of such methods, we examined the roles of environment (i.e., natural selection), random genetic processes (i.e., drift), and demography and life histories (e.g., feeding migrations) on population structure of a widely distributed and abundant marine pelagic fish of economic importance, Atlantic herring (Clupea harengus). Individuals were collected during peak spawning time from 19 spawning locations spanning the region from the western North Sea to the eastern Baltic Sea (N= 1859, eight microsatellite loci). We carried out separate analyses of neutral and selected genetic variation, which allowed us to establish that the two most important factors affecting population structure were selection due to salinity at spawning sites and feeding migrations. The genetic signal left by the demographic history of herring, on the other hand, seems to have been largely eroded, which is not surprising given the large reproductive potential and presumed enormous local effective population sizes of pelagic fish that constrain the effect of stochastic processes. The approach we used can in principle be applied to any abundant and widely distributed aquatic or terrestrial species

A spatio-temporal study of genetic variation in the Danish pine marten (Martes martes) populations from the Jutland peninsula and from the island of Sealand was performed using 11 microsatellite markers. Samples obtained from 1892 to 2003 were subdivided into historical (prior to 1970) and recent (from 1970) groups. As compared with the historical samples, there was a significant loss of genetic variation in the recent Jutland population, but not in Sealand. Effective population sizes were estimated using Bayesian-based software (TMVP). Historical effective population sizes were 5897 (90% highest probability density, HPD, limits: 1502–6849) in Jutland and 1300 (90% HPD limits: 224–5929) in Sealand, whereas recent effective population sizes were 14.7 (90% HPD limits: 10.9–23.5) in Jutland and 802 (90% HPD limits: 51.8–5510) in Sealand. Significant genetic differentiation (F ST ) was found between the two historical samples, between the two recent samples, and between the historical and the recent sample in Jutland; whereas the F ST value between the historical and the recent sample in Sealand was not significant. The significant genetic differentiation between the historical and the recent samples indicates changes in the genetic compositions over time, and the higher F ST values between the two recent samples, as compared with the two historical samples, indicates that the populations in Sealand and Jutland have drifted apart within a short time span. No deviation from Hardy–Weinberg equilibrium was found within populations, indicating no further substructuring

The ability to recognize individual animals has substantially increased our knowledge of the biology and behaviour of many taxa(1). However, not all species lend themselves to this approach, either because of insufficient phenotypic variation or because tag attachment is not feasible. The use of genetic markers ('tags') represents a viable alternative to traditional methods of individual recognition, as they are permanent and exist in all individuals. We tested the use of genetic markers as the primary means of identifying individuals in a study of humpback whales in the North Atlantic Ocean. Analysis of six microsatellite loci(2,3) among 3,060 skin samples collected throughout this ocean allowed the unequivocal identification of individuals. Analysis of 692 'recaptures', identified by their genotype, revealed individual local and migratory movements of up to 10,000 km, limited exchange among summer feeding grounds, and mixing in winter breeding areas, and also allowed the first estimates of animal abundance based solely on genotypic data. Our study demonstrates that genetic tagging is not only feasible, but generates data (for example, on sex) that can be valuable when interpreting-the results of tagging experiments.

Between three and six tri- and tetranucleotide repeat microsatellite loci were analyzed in 3720 samples collected from four different species of baleen whales. Ten of the 18 species/locus combinations had imperfect allele arrays, i.e., some alleles differed in length by other than simple integer multiples of the basic repeat length. The estimate of the average number of alleles and heterozygosity was higher at loci with imperfect allele arrays relative to those with perfect allele arrays. Nucleotide sequences of 23 different alleles at one tetranucleotide repeat microsatellite locus in fin whales, Balaenoptera physalus, and humpback whales, Megaptera novaeangliae, revealed sequence changes including perfect repeats only, multiple repeats, and partial repeats. The relative rate of the latter two categories of mutation was estimated at 0.024 of the mutation rate involving perfect repeats only. It is hypothesized that single-strand slippage of partial repeats may provide a mechanism for counteracting the continuous expansion of microsatellite loci, which is the logical consequence of recent reports demonstrating directional mutations. Partial-repeat mutations introduce imperfections in the repeat array, which subsequently could reduce the rate of single-strand slippage. Limited computer simulations confirmed this predicted effect of partial-repeat mutations.