Articular cartilage gene expression patterns in the tissue surrounding the impact site following applications of shear and axial loads.

BACKGROUND: Osteoarthritis is a degradative joint disease found in humans and commercial swine which can develop from a number of factors, including prior joint trauma. An impact injury model was developed to deliver in vitro loads to disease-free porcine patellae in a model of OA. METHODS: Axial impactions (2000 N normal) and shear impactions (500 N normal with induced shear forces) were delivered to 48 randomly assigned patellae. The patellae were then cultured for 0, 3, 7, or 14 days following the impact. Specimens in the tissue surrounding the loading site were harvested and expression of 18 OA related genes was studied via quantitative PCR. The selected genes were previously identified from published work and fell into four categories: cartilage matrix, degradative enzymes, inflammatory response, and apoptosis. RESULTS: Type II collagen (Col2a1) showed significantly lower expression in shear vs. axial adjacent tissue at day 0 and 7 (fold changes of 0.40 & 0.19, respectively). In addition, higher expression of degradative enzymes and Fas, an apoptosis gene, was observed in the shear specimens. CONCLUSIONS: The results suggest that a more physiologically valid shear load may induce more damage to surrounding articular cartilage than a normal load alone.

The effect of breed and sex on sulfamethazine, enrofloxacin, fenbendazole and flunixin meglumine pharmacokinetic parameters in swine.

Drug use in livestock has received increased attention due to welfare concerns and food safety. Characterizing heterogeneity in the way swine populations respond to drugs could allow for group-specific dose or drug recommendations. Our objective was to determine whether drug clearance differs across genetic backgrounds and sex for sulfamethazine, enrofloxacin, fenbendazole and flunixin meglumine. Two sires from each of four breeds were mated to a common sow population. The nursery pigs generated (n = 114) were utilized in a random crossover design. Drugs were administered intravenously and blood collected a minimum of 10 times over 48 h. A non-compartmental analysis of drug and metabolite plasma concentration vs. time profiles was performed. Within-drug and metabolite analysis of pharmacokinetic parameters included fixed effects of drug administration date, sex and breed of sire. Breed differences existed for flunixin meglumine (P-value<0.05; Cl, Vdss ) and oxfendazole (P-value<0.05, AUC0→∞ ). Sex differences existed for oxfendazole (P-value < 0.05; Tmax ) and sulfamethazine (P-value < 0.05, Cl). Differences in drug clearance were seen, and future work will determine the degree of additive genetic variation utilizing a larger population.

High dietary iron reduces transporters involved in iron and manganese metabolism and increases intestinal permeability in calves.

A 56-d experiment was designed to examine the effect of high dietary Fe on metal transporters involved in Fe and Mn metabolism. Fourteen weaned Holstein calves were stratified by weight and randomly assigned to 1 of 2 treatments: 1) no supplemental Fe (normal Fe) or 2) 750mg of supplemental Fe/kg of dry matter (high Fe). Jugular blood was collected on d 0, 35, and 56. At the end of the trial, 6 calves per treatment were humanely killed and duodenal scrapings, liver, and heart were collected for analysis. Additionally, proximal duodenum was mounted on Ussing chambers to assess intestinal barrier integrity. Calves receiving high dietary Fe displayed decreased transepithelial resistance and increased apical-to-basolateral flux of radiolabeled mannitol, suggesting that high Fe created increased intestinal permeability. Feeding calves a diet high in Fe decreased average daily gain, dry matter intake, and feed efficiency. Hemoglobin and serum Fe concentrations did not differ due to dietary treatment. High dietary Fe increased concentrations of Fe in the liver, but did not affect heart or duodenal Fe concentrations. Duodenal Mn concentrations were lowered by feeding a high Fe diet, but liver and heart Mn concentrations were not affected. As determined by real-time reverse transcription PCR, relative hepatic expression of the gene that encodes the Fe regulatory hormone hepcidin was 5-fold greater in calves fed high dietary Fe. Hepcidin is released in response to increased Fe status and binds to the Fe export protein ferroportin causing ferroportin to be degraded, thereby reducing dietary Fe absorption. Confirmation of this result was achieved through Western blotting of duodenal protein, which revealed that ferroportin was decreased in calves fed high dietary Fe. Duodenal protein expression of divalent metal transporter 1 (DMT1), a Fe import protein that can also transport Mn, tended to be reduced by high dietary Fe. Transcript levels of several genes involved in Fe metabolism in liver and duodenum were unchanged by treatment. In summary, feeding calves a diet high in Fe induced a signal cascade (hepcidin) designed to reduce absorption of Fe (via reduced protein expression of ferroportin and DMT1) in a manner similar to that reported in rodents. Additionally, reduced levels of DMT1 protein appeared to decrease duodenal Mn, suggesting that Mn may also be a substrate for DMT1 in cattle.

Comparative mapping of bovine chromosome 27 with human chromosome 8 near a dairy form QTL in cattle.

In the absence of a complete and annotated bovine genome sequence, detailed human-bovine comparative maps are one of the most effective tools for identification of positional candidate genes contributing to quantitative trait loci (QTL) in cattle. In the present study, eight genes from human chromosome 8 were selected for mapping in cattle to improve breakpoint resolution and confirm gene order on the comparative map near the 40 cM region of the BTA27 linkage map where a QTL affecting dairy form had previously been identified. The resulting map identified ADRB3 as a positional candidate gene for the QTL contributing to the dairy form trait based on its estimated position between 40 and 45 cM on the linkage map. It is also a functional candidate gene due to its role in fat metabolism, and polymorphisms in the ADRB3 gene associated with obesity and metabolic disease in humans, as well as, carcass fat in sheep. Further studies are underway to investigate the existence of polymorphisms in the bovine ADRB3 gene and their association with traits related to fat deposition in cattle.

Detection of quantitative trait loci influencing conformation traits and calving ease in Holstein-Friesian cattle.

An extension of our previous genome scan of a North American Holstein-Friesian population was conducted to identify quantitative trait loci (QTL) affecting conformation traits. Resource families consisted of 1404 sons of 10 elite sires. Genome coverage was estimated to be 2713.5 cM (90%) for 406 markers using a granddaughter design. Regression interval mapping was used to detect QTL affecting 22 conformation traits, including body, udder, feet and legs, and dairy conformation as well as calving ease. Analysis of the families jointly identified 41 chromosome-wise significant QTL influencing conformation traits and 3 significant QTL influencing calving ease on 20 chromosomes. The false discovery rate method was used to account for multiple testing and 3/4 of the suggestive and 5/6 of significant QTL should be real effects. Fourteen of the 44 QTL were significant at the genome-wise level. Comparison of these results with other published reports identifies common QTL affecting conformation traits. Regions on 10 chromosomes appear to affect multiple traits, including conformation, milk production, and somatic cell score, within these particular US Holstein families. Additional work is needed to determine the precise locations of the QTL and select positional candidate genes influencing these traits.

Effect of dietary copper amount and source on copper metabolism and oxidative stress of weanling pigs in short-term feeding.

Forty-eight weanling barrows were used to determine the effects of amount and source of dietary Cu on Cu metabolism, oxidative stress in the duodenum, and VFA ratios in the cecum of weanling pigs in short-term feeding. At 21 d of age, newly weaned pigs were stratified by BW (7.03 ± 1.20 kg) and equally assigned to 1 of the following dietary treatments: 1) control (5 mg supplemental Cu/kg diet from CuSO4), 2) 225 mg supplemental Cu/kg diet from CuSO4, or 3) 225 mg supplemental Cu/kg diet from tribasic Cu chloride (TBCC). Pigs were housed 2 pigs per pen and were fed a complex diet until harvest on d 11 and 12. During harvest, bile and liver were obtained for mineral analysis, and liver samples were obtained for analysis of mRNA expression of Cu regulatory proteins. Digesta of duodenum, proximal jejunum, and ileum were collected for soluble Cu analysis. Mucosal scrapings of duodenum, proximal jejunum, and ileum were obtained for analysis of mucosal Cu concentration and mRNA expression of Cu regulatory proteins. Duodenal mucosal scrapings were also collected for analysis of malondialdehyde (MDA). Pigs fed high Cu had markedly greater (P < 0.0001) Cu concentrations in the duodenal, proximal jejunal, and ileal mucosa than controls. Copper in the duodenal mucosa was greater (P = 0.003) in CuSO4 than TBCC pigs. Duodenal MDA concentrations were greater (P = 0.003) in CuSO4 vs. control pigs and tended (P = 0.06) to be greater than in TBCC pigs. Duodenal antioxidant 1 (Atox1) mRNA was downregulated (P < 0.01) in pigs fed high Cu compared to controls and was not affected by Cu source. Compared with control pigs, those fed CuSO4 and TBCC had greater (P < 0.001) liver and bile Cu concentrations. Liver Cu was also greater (P = 0.0007) in TBCC than CuSO4-fed pigs. Hepatic Cu transporting ß-polypeptide ATPase (Atp7b) was upregulated (P = 0.02) in the Cu-supplemented pigs compared with controls and did not differ among Cu sources. The acetate:propionate ratio in cecal contents was much greater in pigs supplemented with 225 mg Cu/kg diet than in controls. When fed at 225 mg Cu/kg diet, TBCC may cause less oxidative stress in the duodenum than CuSO4. Feeding weanling pigs increased Cu resulted in modulation of duodenal and liver at the transcription level.

Characterization and expression of the bovine growth hormone-releasing hormone (GHRH) receptor.

The hypothalamic hormone, growth hormone-releasing hormone (GHRH) and its pituitary receptor are principal regulators of pituitary growth hormone (GH) synthesis and release. In the present study, we cloned and sequenced a complete bovine pituitary GHRH receptor cDNA in order to study its expression in cattle. The lengths of the exons in the bovine GHRH receptor gene were determined by comparison of the cloned cDNA with genomic sequences obtained from a bovine genomic library clone. As in other species, the bovine cDNA sequence encodes a 423-amino acid protein containing seven hydrophobic domains characteristic of a G protein-coupled receptor. The predicted bovine amino acid sequence shares 93, 90, 89, 87, and 85% identity with the ovine, porcine, human, rat and mouse sequences, respectively. Expression of the receptor in bovine ileum, ovary, anterior pituitary, testis, hypothalamus, pancreas and liver was examined by RT-PCR. Of those tissues examined, GHRH receptor expression was detected in the anterior pituitary gland and hypothalamus. To gain a better understanding of GHRH receptor gene regulation in ruminants, we examined the effect of bovine somatotropin (bST) treatment on pituitary GHRH receptor expression in dairy heifers using relative and real-time RT-PCR. In the present study, bST treatment of dairy heifers resulted in no significant decline in pituitary GHRH receptor expression.

Progression of Gene Expression Changes following a Mechanical Injury to Articular Cartilage as a Model of Early Stage Osteoarthritis.

An impact injury model of early stage osteoarthritis (OA) progression was developed using a mechanical insult to an articular cartilage surface to evaluate differential gene expression changes over time and treatment. Porcine patellae with intact cartilage surfaces were randomized to one of three treatments: nonimpacted control, axial impaction (2000 N), or a shear impaction (500 N axial, with tangential displacement to induce shear forces). After impact, the patellae were returned to culture for 0, 3, 7, or 14 days. At the appropriate time point, RNA was extracted from full-thickness cartilage slices at the impact site. Quantitative real-time PCR was used to evaluate differential gene expression for 18 OA related genes from four categories: cartilage matrix, degradative enzymes and inhibitors, inflammatory response and signaling, and cell apoptosis. The shear impacted specimens were compared to the axial impacted specimens and showed that shear specimens more highly expressed type I collagen (Col1a1) at the early time points. In addition, there was generally elevated expression of degradative enzymes, inflammatory response genes, and apoptosis markers at the early time points. These changes suggest that the more physiologically relevant shear loading may initially be more damaging to the cartilage and induces more repair efforts after loading.

Effect of dietary copper and breed on gene products involved in copper acquisition, distribution, and use in Angus and Simmental cows and fetuses.

Copper (Cu) deficiency is a widespread problem in cattle across the United States and breed differences in Cu metabolism may contribute to this issue. Intracellular Cu is tightly regulated by transport and chaperone proteins, and to date, these mechanisms have not been elucidated to address breed differences in Cu metabolism, nor have these proteins been characterized in bovine fetal liver. Mature, pregnant Angus (n = 8) and Simmental (n = 8) cows (∼4 mo into gestation) were used in a 2 × 2 factorial arrangement of treatments. All cows were bred to Angus sires resulting in an Angus vs. Simmental × Angus comparison for fetuses. Cows were randomly assigned to corn silage-based diets that were either adequate (+Cu) or deficient (-Cu; 6.6 mg Cu/kg DM) in Cu. Diets were individually fed for 112 d. At the end of the study, cows were harvested to collect duodenal mucosa scrapes, liver samples, and fetal liver samples for mineral analysis and also for mRNA and protein analysis of Cu transport and chaperone proteins. Placentomes were also obtained for mineral analysis. Plasma Cu and liver Cu were affected by Cu, breed, and Cu × breed. Both of these Cu indices were less (P ≤ 0.05) in-Cu Simmentals (-CuS) than in-Cu Angus (- uA), but were similar among +Cu Simmental (+CuS) and +Cu Angus cows (+CuA). Duodenal Cu was less (P = 0.01) in-Cu vs. +Cu cows. Placentome Cu was less (P = 0.003) in-Cu vs. +Cu cows, and was also less (P = 0.03) in Simmentals vs. Angus. Fetal liver Cu was less (P = 0.002) in-Cu vs. +Cu fetuses, and was also less (P = 0.05) in Simmental × Angus vs. Angus. Abundance of Cu transporter1 (CTR1) protein and transcripts for Cu transporters and chaperones were not affected by Cu or breed in liver and were not affected by Cu in the intestine. Duodenal Ctr1 was less (P = 0.04) and CTR1 tended (P = 0.10) to be less in Simmentals vs. Angus. Expression of Atp7a tended (P = 0.08) to be less in Simmentals than in Angus. In fetal liver, expression of antioxidant 1 (Atox1), cytochrome c oxidase assembly protein 17 (Cox17), and Cu metabolism MURR1 domain 1 (Commd1) were up-regulated (P ≤ 0.05) in-Cu vs. +Cu fetuses. In conclusion, less expression of duodenal Ctr1 and a tendency for less CTR1 (P = 0.10) and Atp7a (P = 0.08) suggest that Simmentals have a lesser ability to absorb and utilize dietary Cu, and may explain why Simmentals are more prone to Cu deficiency than Angus. Up-regulation of fetal liver Atox1, Cox17, and Commd1 in-Cu fetuses may reflect the great Cu demand by the fetus.

Amount and source of dietary copper affects small intestine morphology, duodenal lipid peroxidation, hepatic oxidative stress,and mRNA expression of hepatic copper regulatory proteins in weanling pigs.

Thirty weanling, crossbred barrows (SUS SCROFA) were used to determine the effects of amount and source of dietary Cu on small intestinal morphology and lipid peroxidation, Cu metabolism, and mRNA expression of proteins involved in hepatic Cu homeostasis. At 21 d of age, pigs were stratified by BW (6.33 ± 0.23 kg) and allocated to 1 of the following dietary treatments: i) control (no supplemental Cu; 6.7 mg Cu/kg), ii) 225 mg supplemental Cu/kg diet from Cu sulfate (CuSO(4)), or iii) 225 mg supplemental Cu/kg diet from tribasic Cu chloride (TBCC). Pigs were housed 2 pigs per pen and were fed a 3-phase diet regimen until d 35 or 36 of the study. During harvest, bile and liver were obtained for mineral analysis, and liver samples were also obtained for analysis of liver glutathione (GSH) and mRNA expression of Cu regulatory proteins. Segments of duodenum, proximal jejunum, and ileum were obtained for mucosal morphology, and duodenal mucosal scrapings were collected from all pigs for analysis of malondialdehyde (MDA). Duodenal villus height was reduced in CuSO(4) pigs compared with control (P = 0.001) and TBCC (P = 0.03) pigs. Villus height in the proximal jejunum of CuSO(4) pigs was reduced (P = 0.03) compared with control pigs, but ileal villus height was not affected (P = 0.82) by treatment. Duodenal MDA concentrations were greater (P = 0.03) in CuSO(4) pigs and tended to be greater (P = 0.10) in pigs supplemented with TBCC compared with control pigs. Liver Cu was greater (P = 0.01) in CuSO(4) vs. control pigs, and tended (P = 0.07) to be greater in TBCC pigs than control pigs. Bile Cu concentrations were greater (P < 0.001) in CuSO(4) and TBCC pigs vs. controls and were also greater (P = 0.04) in TBCC vs. CuSO(4) pigs. Total liver GSH concentrations were less (P = 0.02) in pigs fed diets supplemented with CuSO(4) vs. pigs fed control diets but total liver GSH did not differ (P = 0.11) between control and TBCC pigs. Hepatic mRNA of cytochrome c oxidase assembly protein 17 was less (P = 0.01) in CuSO(4) and tended to be less (P = 0.08) in TBCC pigs vs. control pigs. Expression of antioxidant 1 mRNA was greater (P = 0.04) in TBCC pigs and tended to be greater (P = 0.06) in CuSO(4) pigs compared with control pigs. Results of this study indicated that, when fed at 225 mg Cu/kg diet, TBCC may cause less oxidative stress in the duodenum than CuSO(4). Feeding weanling pigs increased Cu resulted in modulation of certain Cu transporters and chaperones at the transcription level.

Investigation of breed and sex effects on cytochrome P450 gene expression in cattle liver.

Many cytochrome P450 enzymes are involved in xenobiotic metabolism and elimination. In humans, genetic variation in some of these enzymes contributes to inter-individual drug responses, sometimes having significant clinical effects. Transcript levels of eight P450 genes were evaluated in liver to investigate potential differences in breed and sex in cattle. In Angus calves, heifers appeared to have higher gene expression than steers for two of the eight genes. In Angus and Simmental pregnant cows, Angus appeared to have higher gene expression for three of the eight genes. Transcript evaluation is just the first step toward determining if differences exist between breeds and sexes in enzyme catalytic activity. However, others (Giantin et al., 2008) have shown correlations between transcript levels and catalytic activity in other cattle breeds. Therefore breed and/or sex of an animal may need to be considered before administering a dose of a xenobiotic due to the potential for harmful drug residues in foodstuffs as well as improper treatment of disease conditions.

Proteins involved in iron metabolism in beef cattle are affected by copper deficiency in combination with high dietary manganese, but not by copper deficiency alone.

A 493-d study was conducted to determine the impact of a severe, long-term Cu deficiency on Fe metabolism in beef cattle. Twenty-one Angus calves were born to cows receiving one of the following treatments: 1) adequate Cu (+Cu), 2) Cu deficient (-Cu), and 3) Cu deficient plus high Mn (-Cu+Mn). Copper deficiency was induced through the addition of 2 mg of Mo/kg of DM. After weaning, calves remained on the same treatment as their dam through growing (basal diet analyzed 7 mg of Cu/kg of DM) and finishing (analyzed 4 mg of Cu/kg of DM) phases. Plasma Fe concentrations were positively correlated (P < 0.01; r = 0.49) with plasma Cu concentrations. Liver Fe concentrations were greater (P = 0.05) in -Cu vs. +Cu calves and further increased (P = 0.07) in -Cu+Mn vs. -Cu calves. There was a negative relationship (P < 0.01; r = -0.31) between liver Cu and Fe concentrations. This relationship is likely explained by less (P < 0.01) plasma ceruloplasmin activity in -Cu than +Cu calves. As determined by real-time reverse transcription-PCR, relative expression of hepatic hepcidin was significantly downregulated (>1.5 fold) in -Cu compared with +Cu calves (P = 0.03), and expression of hepatic ferroportin tended (P = 0.09) to be downregulated in -Cu vs. +Cu. In the duodenum, ferritin tended to be upregulated in -Cu. vs. +Cu calves (P < 0.06). No significant change (P > 0.2) due to Cu-deficiency was detected at the transcriptional level for either isoform of divalent metal transporter 1 (DMT1 mRNA with or without an iron responsive element; dmt1IRE and dmt1-nonIRE) in liver or intestine. Duodenal expression of hephaestin and ferroportin protein was not affected by dietary treatment (P > 0.20). However, duodenal expression of DMT1 protein was less (P = 0.04) in -Cu+Mn steers vs. -Cu steers. In summary, Cu deficiency alone did affect hepatic gene expression of hepcidin and ferroportin, but did not affect duodenal expression of proteins important in Fe metabolism. However, the addition of 500 mg of Mn/kg of DM to a diet low in Cu reduced duodenal expression of the Fe import protein DMT1.

Gene expression profiling of chondrocytes from a porcine impact injury model.

OBJECTIVE: To identify differentially expressed genes between axially impacted and control articular cartilage taken from porcine patellae maintained in organ culture for 14 days. METHODS: Porcine patellae were impacted perpendicular to the articular surface to create an impact injury. Intact patellae (control and impacted) were maintained in culture for 14 days. Total RNA was then extracted from the articular cartilage beneath the impaction and used to prepare two Serial Analysis of Gene Expression (SAGE) libraries. Approximately 42,500 SAGE long tags were sequenced from the libraries. The expression of select genes was confirmed by quantitative real-time PCR analysis. RESULTS: Thirty-nine SAGE tags were significantly differentially expressed in the impacted and control libraries, representing 30 different annotated pig genes. These genes represented gene products associated with matrix molecules, iron and phosphate transport, protein biosynthesis, skeletal development, cell proliferation, lipid metabolism and the inflammatory response. Twenty-three of the 30 genes were down-regulated in the impacted library and five were up-regulated in the impacted library. Quantitative real-time PCR follow-up of four genes supported the results found with SAGE. CONCLUSION: We have identified 30 putative genes differentially expressed in a porcine impact injury model and validated these findings for four of these genes using real-time PCR. Results using this impact injury model have contributed further evidence that damaged chondrocytes may de-differentiate into fibroblast-like cells and proliferate in an attempt to repair themselves. Additional work is underway to study these genes in further detail at earlier time points to provide a more complete story about the fate of chondrocytes in articular cartilage following an injury.

Refinement of quantitative trait loci on bovine chromosome 18 affecting health and reproduction in US Holsteins.

Selection for increased milk yield is associated with decreased fertility in US Holsteins. Previously, a putative quantitative trait locus (QTL) on chromosome 18 affecting daughter pregnancy rate (DPR) was identified in one family. Our aim was to determine the validity of the QTL using additional markers and an extended pedigree. Twelve markers were genotyped in 940 descendants of the original sire in which the QTL was identified. Analysis of the extended pedigree detected significant and suggestive QTL for DPR, productive life and somatic cell score. Further analysis is underway to refine the QTL region so that positional candidate genes can be identified.

Characterization of a line of pigs previously selected for increased litter size for RBP4 and follistatin.

The objective of this study was to determine if selection response for increased litter size in pigs could be partially attributed to three type 1 marker loci coding for genes known to affect litter size: oestrogen receptor (ESR), retinol-binding protein 4 (RBP4) and follistatin (FS). In the high litter size line (LS), pigs from the largest litters, based on number of pigs born alive (NBA), were retained to parent the next generation. A randomly selected control line (LC) was maintained. Gilts were reared in litters of 10 pigs or less to minimize maternal effects. Pigs were measured at generations 10-12. Additional traits scored were number of fully formed pigs (NFF) and number of mummified fetuses (MUM). Breeding values for NFF and NBA were greater (p < 0.05) in LS than LC in generations 11 and 12, but no significant line differences were found for MUM. The A allele of the ESR locus was fixed in both lines. After adjustment for effects of genetic drift, frequency of the two alleles segregating for the FS and RBP4 loci did not differ significantly between lines. No significant additive or dominance effects of the FS markers were detected for NFF, NBA and MUM in either LS or LC. Response to selection for increased litter size could not be attributed to effects at the ESR, RBP4 or FS loci.

Whole-genome scan to detect QTL for milk production, conformation, fertility and functional traits in two US Holstein families.

A genome scan was conducted in two US Holstein half-sib families to identify quantitative trait loci (QTL) affecting milk production and conformation traits using the granddaughter design. The sires of the two studied families were related as sire and son and had 96 and 212 sons respectively. A total of 221 microsatellite loci were scored in both families. Statistical analysis was performed using two different analytical methods; half-sib least squares regression and Bayesian Monte Carlo Markov Chain. Traits analysed included five traditional milk production traits, somatic cell count, daughter pregnancy rate, male fertility and 20 conformation traits. A total of 47 tests achieved at least genome-wise significance. However, results from the two methods of analysis were only concordant for QTL location and level of significance in eight instances.

The bovine butyrophilin gene maps to chromosome 23.

Chromosomal assignment of the bovine butyrophilin gene (BTN) was performed by analysis of DNA from somatic hybrid cell lines using the polymerase chain reaction. The gene was assigned to bovine chromosome 23 using two sets of primers specific for bovine BTN.

Detection of putative loci affecting milk, health, and type traits in a US Holstein population using 70 microsatellite markers in a genome scan.

Quantitative trait loci affecting milk yield, health, and type traits were studied for seven large U.S. Holstein grandsire families using the granddaughter design. Seventy microsatellite markers located throughout the genome were selected for the quantitative trait loci scan. Effects of marker alleles were analyzed for 30 traits (21 type traits, 5 milk traits, 2 calving ease triats, and somatic cell score and productive life) and 16 canonical traits derived from type and production traits. Previously we reported results from 36 of these markers but have re-evaluated those results using a more robust analysis method. We report results from all 70 markers using permutation tests to calculate experiment-wise significance values, replacing the less stringent comparison-wise values previously reported. With this new methodology we detected 9 putative quantitative trait loci within specific families. Four markers were associated with effects on type traits on chromosomes 4, 5, 14, and 23. Two markers were associated with effects on protein percentage on chromosomes 6 and 14, and 3 markers were associated with effects on productive life on chromosomes 2, 21, and 23. Although these initial 70 microsatellite markers have been completed in the 7 Holstein families, additional markers will need to be added to allow interval analysis of areas where putative QTL have been identified and to increase marker density where needed.

Genetic diversity and background linkage disequilibrium in the North American Holstein cattle population.

The objectives of this study were to 1) identify highly heterozygous Holstein bulls that are as unrelated as possible and widely used in the US dairy industry; 2) quantify the level of genetic diversity in US Holsteins; and 3) determine the extent of background linkage disequilibrium (BLD) and disease trait associated linkage disequilibrium (DLD) in the US Holstein population. Twenty-three Holstein bulls that are not closely related but were widely used in the US dairy industry were genotyped for 54 microsatellite loci. The genotyping was performed on automated DNA sequencers (PE Applied Biosystems, CA), following polymerase chain reaction amplification with fluorescent dye-labeled primers. The heterozygosity for the sampled population ranged from 0.43 to 0.80. This wide range of heterozygosity allows selection of the most heterozygous bulls to develop informative families for gene mapping studies. The degree of genetic diversity in this population is significant and allows selection for traits of economic importance. As expected, there is extensive linkage disequilibrium (LD) in the US Holstein population. About half of the syntenic marker pairs presented a typical pattern of LD produced by DLD. Most of the nonsyntenic marker pairs had a typical pattern of LD arising from BLD. These results suggest that the observed LD is not purely due to genetic drift and migration and that a portion might be due to DLD. This raises our hopes of successful fine-localization of genes for complex traits using LD mapping.