Progress 10/01/08 to 09/30/13
Outputs
Target Audience: Scientists working in similar research areas through publications in peer reviewed journals Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Two graduate students and one post-doctoral fellow were trained during this project How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
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. 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.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
(1) Cabrera, R., Lin, X., Ashwell, M., Moeser, A., and Odle, J. (2013) Early post-natal kinetics of colostral IgG absorption in fed and fasted piglets and developmental expression of the intestinal IgG receptor. Journal of Animal Science, in press.
(2) Fry, R.S., Ashwell, M.S., Engle, T.E., Han, H., Hansen, S.L. and Spears, J.W. (2013) Effect of dietary copper and breed on gene products involved in copper acquisition, distribution, and utilization in Angus and Simmental cows and their fetuses. Journal of Animal Science. 91:861-71.
(3) Ashwell, M.S., Gonda, M.G., Maltecca, C., Gray, K.A. ONan, A.T., Cassady, J.P., and Mente, P.L. (2013) Changes in chondrocyte gene expression following in vitro impaction of porcine articular cartilage in an impact injury model. Journal of Orthopaedic Research. 31: 385-91.
|
Progress 10/01/11 to 09/30/12
Outputs
OUTPUTS: Porcine patellae are being used as a biomedical model for development of osteoarthritis after an impact injury. Expression levels for genes of interest must be normalized with an appropriate reference, or housekeeping gene, to make accurate comparisons of quantitative real-time PCR results. We identified the most stable housekeeping genes in porcine articular cartilage subjected to a mechanical injury from a panel of 10 candidate genes. Ten housekeeping genes were evaluated in three different treatment groups of mechanically impacted porcine articular cartilage. The stability of the genes was measured using geNorm, BestKeeper, and NormFinder software. The three programs generated similar results for the most stable genes in porcine articular cartilage, which were succinate dehydrogenase flavoprotein, subunit A, peptidylprolyl isomerase A, glyceraldehyde-3-phosphate dehydrogenase, beta actin. Using our stable housekeeping gene results, we monitored chondrocyte gene expression at 0, 3, 7, and 14 days following in vitro impaction to the articular surface of porcine patellae. Patellar facets were either axially impacted with a cylindrical impactor at 25 mm per s loading rate to a load level of 2,000 newtons or not impacted to serve as controls. After being placed in organ culture for 0, 3, 7, or 14 days, total RNA was isolated from full thickness cartilage slices and gene expression measured for 17 genes by quantitative real-time RT-PCR. Targeted genes included those encoding proteins involved with biological stress, inflammation, or anabolism and catabolism of cartilage extracellular matrix. Some gene expression changes were detected on the day of impaction, but most significant changes occurred at 14 days in culture. At 14 days in culture, 10 of the 17 genes were differentially expressed with col1a1 most significantly up-regulated in the impacted samples. Other work examined changes in liver gene expression when the amount and source of dietary copper was changed in weanling barrows. Thirty weanling, crossbred barrows were used to determine the effects of amount and source of dietary Cu on small intestinal mRNA expression of proteins involved in hepatic Cu homeostasis. At 21 d of age, pigs were stratified by body weight and allocated to one of the following dietary treatments: i) control (no supplemental Cu), ii) 225 mg supplemental Cu per kg diet from Cu sulfate (CuSO4), or iii) 225 mg supplemental Cu per 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, liver samples were obtained for analysis of mRNA expression of Cu regulatory proteins. Hepatic mRNA of cytochrome c oxidase assembly protein 17 was less in CuSO4 and tended to be less in TBCC pigs vs. control pigs. Expression of antioxidant 1 mRNA was greater in TBCC pigs and tended to be greater in CuSO4 pigs compared with control pigs. PARTICIPANTS: Melissa Ashwell, principal investigator Robert Fry, graduate student Jerry Spears, principal investigator Audrey ONan, technician Karen Lloyd, technician Michael Gonda, post doctoral fellow Peter Mente, principal investigator Christian Maltecca, investigator TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Expression levels for genes of interest must be normalized with an appropriate reference, or housekeeping gene, to make accurate comparisons of quantitative real-time PCR results, therefore we needed to determine which genes were the most stably expressed to accurately evaluate the changes in expression levels of genes we believe are involved in degenerative joint disease progression. Using the results from the housekeeping study allowed us to study 17 experimental genes. From that study we determined that 10 of the 17 genes were differentially expressed after 14 days in culture. We found col1a1 most significantly up-regulated in the impacted samples suggesting impacted chondrocytes may have reverted to a fibroblast-like phenotype. These data agree with other published work in this area conducted by us as well as others throughout the world.Understanding the progression of changes in naturally occurring OA will help us understand the degenerative process and determine methods to prevent or even reverse the degeneration. The development of an in vitro model that mimics these changes will provide a test bed to examine potential treatments and help increase our understanding of the complex interactions between the chondrocytes and matrix that result in tissue degeneration. While OA is a disease that can be slow to develop (often taking years before clinically significant symptoms arise), in an injury model the events that start the tissue down the degenerative pathway presumably occur at the time of injury. Being able to identify what these very early changes are and see how they progress over time provide insight into treatment methods that can be applied to patients following an injury and potentially those with other etiologies that nonetheless follow the same degenerative pathway. Previous studies found that level and source of dietary copper affects swine growth and other physiological parameters. Therefore our study examining different levels and sources of copper attempted to explain some of those differences at the molecular level. Results of this study indicated that feeding weanling pigs increased Cu resulted in modulation of certain Cu transporters and chaperones at the transcription level.
Publications
- McCulloch, R.S., Ashwell, M.S., ONan, A.T., and Mente, P.L. (2012) Identification of stable normalization genes for quantitative real-time PCR in porcine articular cartilage. Journal of Animal Science and Biotechnology. 3:36.
- Cabrera, R., Lin, X., Ashwell, M., Moeser, A., and Odle, J. (2012) Early post-natal kinetics of colostral IgG absorption in fed and fasted piglets and developmental expression of the intestinal IgG receptor. Journal of Animal Science, in press.
- Fry, R.S., Ashwell, M.S., Engle, T.E., Han, H., Hansen, S.L., and Spears, J.W. (2012) Effect of dietary copper and breed on gene products involved in copper acquisition, distribution, and utilization in Angus and Simmental cows and their fetuses. Journal of Animal Science, in press.
- Fry, R.S., Ashwell, M.S., Lloyd, K.E., ONan, A.T., Flowers, W.L., Stewart, K.R., and Spears, J.W. (2012) 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. Journal of Animal Science, in press.
- Ashwell, M.S., Gonda, M.G., Maltecca, C., Gray, K.A., ONan, A.T., Cassady, J.P., and Mente, P.L. (2012) Changes in chondrocyte gene expression following in vitro impaction of porcine articular cartilage in an impact injury model. Journal of Orthopaedic Research, in press.
|
Progress 10/01/09 to 09/30/10
Outputs
OUTPUTS: Identifying gene expression changes following a traumatic joint injury may help our understanding of early stage osteoarthritis in humans and animals and identify targets for future intervention to slow disease progression. Our objective was to examine gene expression changes for genes implicated in OA following two types of impact injuries; an axial impaction with loading normal to the cartilage surface and a shear impaction that introduces significant tangential loading. Quantitative real-time PCR was used to measure expression levels of 18 genes in 72 porcine patellae. Current results indicate that shear impactions showed an increase in degenerative gene expression soon after the impact injury but tended to express more normal cartilage matrix products 14 days after injury, indicating the potential for a more robust repair process in the shear samples compared to the axial samples. Further work is underway to also examine differences in protein expression in these same samples to compare gene expression profiles with protein expression profiles. Copper is an essential mineral but must be tightly regulated to prevent deficiency or toxicity. It has been well documented that the metabolism of iron and copper are closely related. The effect of dietary iron on copper transporters and chaperones has not been evaluated in the porcine model, nor has these mechanisms been identified in the pig. Therefore weanling male pigs were used to characterize copper transporters and chaperones in the pig at different dietary levels of iron to provide a better understanding of the interactions that occur at the cellular level between iron and copper. In addition, previous studies have shown that level and source of dietary copper affects pig performance. Therefore a study was conducted to determine the effects of level and source of dietary copper on copper concentration and mRNA expression of copper regulatory proteins in the liver and small intestine of weanling pigs. Two sources of copper were fed; copper sulfate and tribasic copper chloride. Both copper level and source affected uptake, mRNA expression of copper regulatory proteins, morphology and lipid peroxidation throughout the small intestine of the weanling pigs. PARTICIPANTS: M Ashwell, R Fry, J Spears, W Flowers, S Hansen, HC Liu, A Moeser, R McCulloch, A ONan, P Mente, C Maltecca, M Knutson, N Trakooljul, J Hicks, E McKenney, S McLeod, V Fellner TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Results from the osteoarthritis research indicate that gene expression is affected very soon after an impact injury and has a lasting effect. If we can identify targets for future intervention, we may be able to slow or prevent osteoarthritic disease progression. Because these studies are conducted in pigs, these results serve a dual benefit, potentially improving human health as well as the welfare of commercial pigs. Results from the copper research indicate that the source of copper affects small intestine morphology, where some sources of copper potentially cause more oxidative damage than others. Future research may help producers decide which source and level of this important mineral should be provided for maximum benefit to livestock.
Publications
- Ashwell, M.S., Fry, R.S., Spears, J.W., ONan, A.T., and Maltecca, C. (2010) Investigation of breed and sex effects on cytochrome P450 gene expression in cattle liver. Research in Veterinary Science, in press.
- Fry, R.S., Ashwell, M.S., and Spears, J.W. (2010) Duodenal copper transporters in cattle are affected by breed. Journal of Animal Science 88 (Suppl. 2):11.
- McKenney, E., Ashwell, M., ONan, A., McLeod, S., Fellner, V. (2010) The effect of diet on fermentation and microbial strategies in primate and carnivore species. Proceedings of the Sixth Crissey Zoological Nutrition Symposium, December 2010, Raleigh, NC, pp. 41-42.
- McKenney, E.A., ONan, A.T., Fellner, V., and Ashwell, M.S. (2010) Evaluating the QiaAMP DNA stool kit for metagenomic studies in Arctictis binturong. Proceedings of the Eighth Comparative Nutrition Society Symposium, August 2010, Tucson, AZ, pp. 126-130.
- Fry, R.S., Ashwell, M.S., Flowers, W.L., Stewart, K.R., and Spears, J.W. (2010) Effect of level and source of dietary copper on copper metabolism in the small intestine of weanling pigs. Journal of Animal Science 88(Suppl. 1): 499.
- Fry, R.S., Spears, J.W., Hansen, S.L., and Ashwell, M.S. (2010) Dietary iron affects mRNA expression of porcine copper transporters. FASEB Journal. 24:229.7.
- Fry, R.S., Spears, J.W., Hansen, S.L., Liu, H.C., and Ashwell, M.S. (2010) Effects of dietary iron and age on cellular copper metabolism in liver of weanling pigs. Journal of Animal Science 88 (Suppl. 1): 499.
- McCulloch, R.S., Ashwell, M.S., ONan, A.T., and Mente, P.L. (2010) Differential gene expression of chrondocytes from a porcine impact injury model using suitable reference genes. Transactions of the 56th annual meeting of the Orthopedic Research Society.
- McCulloch, R.S., Ashwell, M.S., ONan, A.T., Maltecca, C., and Mente, P.L. (2011) Articular cartilage chondrocyte gene expression alterations following shear and axial impactions. Accepted 57th annual meeting of the Orthopedic Research Society.
|
Progress 10/01/08 to 09/30/09
Outputs
OUTPUTS: Transcript levels of eight P450 genes were evaluated in cattle liver to determine if breed and sex differences exist. In Angus calves, heifers had higher gene expression than steers for 7 of the 8 genes. In Angus and Simmental pregnant cows, Angus had higher gene expression for 4 of the 8 genes. Injury to articular joint cartilage is known to be a risk factor for the development of Osteoarthritis (OA). This study evaluated changes in gene expression following an impact injury to porcine articular cartilage. Porcine patellae were impacted either axially (2000N) or with a shear impaction (500N axial load followed by a lateral) in a hydraulic load frame. Impacted and non-impacted control specimens were cultured for either 3 or 14 days. RNA was isolated from full thickness cartilage slices collected from the area under the impaction site and gene expression was measured using quantitative realtime RT-PCR. Ten genes associated with cell proliferation and apoptosis, collagen type, degradative enzymes, and biological stress were analyzed in 26 specimens. The differences between the two impaction methods were most prominent at the 14 day culture period, suggesting that evaluation of more genes and specimens at this time will help identify effects of a shear vs. axial loading. To investigate effects of testosterone, Duroc boars were divergently selected for 10 generations for both high and low levels of testosterone and then maintained until generation 21 by random mating within lines. Gene expression differences in the 150 and 180 day samples were used for microarray analysis using the 5 individuals within each group and age. Approximately 100 genes with divergent expression patterns in the individuals were identified and selected for further investigation. Additional analysis included validation with Real-Time PCR and Genego pathway analysis to identify biological pathways associated with testosterone. PARTICIPANTS: Melissa Ashwell Joseph Cassady Christian Maltecca Jerry Spears Stephanie Hansen Peter Mente Audrey ONan Robert Fry Dana Stanley Ryan McCulloch TARGET AUDIENCES: Laboratory instruction outreach medical community PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Results from examination of the P450 gene expression in different cattle breeds and between the sexes indicated that significant differences exist between breed and sex. Therefore, administering a standard dose of a xenobiotic without considering the breed and/or sex of the individual needing treatment may lead to harmful drug residues in foodstuffs and improper disease treatments. Results from the testosterone study indicate that we have been able to identify specific genes affected by testosterone that may be able to improve health status, fertility, meat quality, and maturation in boars, and have gained a broader insight into the effects of gene pathways involved in the biological processes affected. Using our cartilage samples we have been able to identify a set of stable housekeeping genes that can be used for reliable real-time PCR results in the pig.
Publications
- Hansen, S.L., Ashwell, M.S., Legleiter, L.R., Fry, R.S., Lloyd, K.E., and Spears, J.W. (2009) The addition of high manganese to a copper deficient diet further depresses copper status and growth of cattle. British Journal of Nutrition. 101:1068-78.
- Ashwell, M.S., Ceddia, R.P., House, R.L., Cassady, J.P., Eisen, E.J., Eling, T.E., Collins, J.B., Grissom, S.F., and Odle, J. (2009) Trans-10, cis-12-conjugated linoleic acid alters hepatic gene expression in a polygenic obese line of mice displaying hepatic lipidosis. Journal of Nutritional Biochemistry, available online doi:10.1016/j.jnutbio.2009.06.013.
- Hansen, S.L., Trakooljul, N., Liu, H.-C.S., Hicks, J.A., Ashwell, M.S. and Spears, J.W. (2009) 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. Journal of Animal Science, in press.
- Hansen, S.L., Ashwell, M.S., Moeser, A.J., Fry, R.S., Knutson, M.D., and Spears, J.W. (2009) High dietary iron reduces transporters involved in iron and manganese metabolism and increases intestinal permeability in calves. Journal of Dairy Science, in press.
|
Progress 10/01/07 to 09/30/08
Outputs
OUTPUTS: Joint injury is a risk factor for the development of degenerative joint diseases such as osteoarthritis (OA). An understanding of the early changes in chondrocyte gene expression following cartilage injury will lead to a better understanding of the degenerative process. Therefore, we wanted to monitor changes in chondrocyte gene expression at 0, 3, 7, and 14 days following an in vitro impaction to the articular surface of porcine patellae. Porcine patellae were axially impacted and cultured for 0, 3, 7, or 14 days. After culture, RNA was isolated from articular cartilage and gene expression was measured by quantitative real-time RT-PCR. We measured expression levels for 16 genes. Of course, transcript levels do not always correlate with protein levels and provide no information on post-translational modifications. Therefore evaluation of proteins is a valuable complement to the gene expression studies. We have isolated total protein from cartilage located on the articular surface of porcine patellae. In this study we are examining differences in chondrocyte protein profiles from impacted and non-impacted cartilage 0, 1, and 3 days post impaction. Samples are being collected and protein isolated from samples for 2-D gel electrophoresis, followed by mass spectrometry of differentially expressed proteins. Other swine projects have involved transcriptional profiling of testicular tissue collected from boars divergently selected for testosterone levels, collected at 150 and 180 days of age. The new pig oligonucleotide microarrays were used to examine differences between ages and genetic lines in these animals. A study was conducted to evaluate the effect of long-term Cu deficiency, either alone or in the presence of high Mn, on growth and Cu status of beef cattle. Twenty-one Angus calves were born to cows that had been on one of the following treatments for at least 410 d by calving: 1) supplemental Cu (+Cu), 2) no supplemental Cu and (-Cu), and 3) -Cu diet plus supplemental Mn (-Cu+Mn). Plasma Cu levels, liver Cu concentrations, weight, and gene expression were examined in these animals. Selection for increased milk yield is associated with decreased fertility in US Holsteins. A putative quantitative trait locus on chromosome 18 affecting daughter pregnancy rate was previously identified in one prominent family. Our aims were to refine the QTL region using additional markers and an extended pedigree, improve our Windows QTL Cartographer for within-family analysis and develop a new mixed model based approach for the across-family analysis. PARTICIPANTS: Melissa Ashwell Joseph Cassady Jerry Spears Stephanie Hansen Peter Mente Zhao-Bang Zeng Xiaohua Gong Audrey ONan Michael Gonda Scott Fry Dana Stanley TARGET AUDIENCES: Laboratory instruction outreach medical community PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
For the porcine cartilage gene expression project, Clu was down-regulated in impacted samples relative to non-impacted samples, and Capns1 and Mmp-3 tended to be up-regulated at 0 days in culture. At 7 days, Col1a1 was up-regulated in impacted samples and remained up-regulated at 14 days. Between days 7 and 14, Chi3l1, Clu, Ctsb, Cxcl16, Fth-1, Ftl, Mmp-1, Mmp-3, and Timp-3 were up-regulated in non-impacted samples. In impacted samples, these genes were either down-regulated, or were not differentially expressed. Many of the genes that were up-regulated in non-impacted samples between days 7 and 14 are involved with various responses to cellular stresses. We believe that these genes were up-regulated in response to the in vitro environment, including the resulting reduced loads on the cartilage tissue. These genes were not up-regulated in impacted samples, suggesting that the impaction either abrogated or delayed the chondrocyte's response to these factors. Increased expression of Col1a1 in impacted samples also suggested that chondrocytes were reverting to a more fibroblast-like phenotype. Changes in expression of genes coding for matrix degrading proteins at 14 days may reflect an attempt to preserve the extra-cellular matrix, though genes coding for both Mmps and Timps were all down-regulated. For the testosterone microarray project, approximately 80 genes were differentially expressed and are being validated using quantitative real-time PCR. For the beef cattle Cu experiment, plasma Cu levels were lower in -Cu calves compared to +Cu calves while high dietary Mn further depressed plasma Cu. Liver Cu concentrations in +Cu calves were greater than in -Cu calves, with no differences between -Cu and -Cu+Mn calves. -Cu+Mn calves gained less than -Cu calves during the growing phase. Dry matter intake during the growing phase did not differ among +Cu and -Cu calves; however, intake was lower in -Cu+Mn calves vs. -Cu calves. Gene expression for the cytochrome c oxidase 1 gene was lower in animals that received the -Cu diet compared with those that received either the +Cu diet or the -Cu+Mn diet. There was a tendency for the superoxide dismutase 1 gene to be down-regulated in the animals receiving the -Cu diet relative to those fed the +Cu or -Cu+Mn diets. In conclusion, feeding a Cu deficient diet in combination with high Mn negatively impacted growth and Cu status of beef cattle. For the dairy cattle QTL project, analysis of the extended dataset detected significant and suggestive QTL for DPR, productive life, somatic cell score and other milk yield traits. These QTL are consistent with previously reported QTL but with narrower QTL support intervals that contain fewer positional candidates. In addition, QTL Cartographer was upgraded to include within-family analysis, and our new mixed model based approach for across-family analysis was implemented.
Publications
- Fry, R.S., Ashwell, M.S., Hansen, S.L., Engle, T.E., Han, H., and Spears, J.W. (2008) Effects of long term copper deficiency on gene profiles of copper transporters and chaperones in the liver of cattle. Proceedings of the 13th International Symposium on Trace Elements in Man and Animals, Pucon, Chile.
- Hansen, S.L., Ashwell, M.S., Legleiter, L.R., Fry, R.S., Lloyd, K.E. and Spears, J.W. (2008) The addition of high manganese to a copper deficient diet further depresses copper status and growth of cattle British Journal of Nutrition, September 8: 1-11.
- Ashwell, M.S., ONan, A.T., Gonda, M.G. and Mente, P.L. (2008) Gene expression profiling of chondrocytes from a porcine impact injury model. Osteoarthritis and Cartilage 16: 936-946.
- Ashwell, M.S., Gonda, M.G., and Mente, P.L. (2008) Changes in chondrocyte transcript profiles in a porcine impact injury model of osteoarthritis. Proceedings of the Swine as a Biomedical Model Conference, p. 25.
|
Progress 10/01/06 to 09/30/07
Outputs
OUTPUTS: Previous studies have indicated that udder conformation traits are correlated with mastitis incidence in dairy cattle. Previous genome scans identified QTL affecting SCS and several udder conformation traits on BTA15. Each QTL from these scans was placed between 34-55 cM on the genetic map. We were interested in determining if one QTL is responsible for the effects on udder conformation and somatic cell score. Thirteen half-sib families were selected for this study in which eleven DNA markers were genotyped. Regression interval mapping and permutation testing were used to detect across and within family QTL for SCS and 22 conformation traits. Significant and suggested QTL exceeded a chromosome-wise P value of 0.01 and 0.05, respectively were detected. Across family analysis revealed a significant QTL for SCS at 27 cM and thurl width at 96 cM. Further analysis is underway to construct haplotypes and further refine the location of the QTL. Examination of the bovine genome
sequence identifies several genes in QTL region associated with immunity. After further refinement of the QTL locations, SNPs in candidate genes will be evaluated in this complex pedigree to determine if they explain the observed effects on SCS and conformation traits. Continued genetic improvement for milk production has been associated with decreased fertility in US Holstein cattle. A previous study identified a putative QTL affecting daughter pregnancy rate (DPR) at 54 cM on chromosome 18 in one Holstein grandsire family. We are currently involved in further refinement of the QTL to identify the DNA sequence responsible for the observed effect. Twenty five microsatellite markers from BTA18, with a concentration of markers between 30-90 cM on the map, have been genotyped in 973 animals that were descendants of the original grandsire in which the QTL was identified. Sons of the grandsire as well as six grandson and six great-grandson families of the original grandsire were genotyped
in this complex pedigree. In a joint analysis of thirteen of the largest families, each containing 10 or more sons, a significant QTL for DPR was detected at 27 cM. QTL affecting DPR was detected in two additional sub-families (Family II-5 and Family III-2), indicating a putative QTL affecting this trait is most likely segregating within this pedigree. Across-family analysis also detected putative QTL on chromosome 18 affecting productive life (PL) at 35 cM, somatic cell score (SCS) at 33 cM and percent difficult births at 72 cM (Figure 1B). The approach used to date has produced very large confidence intervals, preventing further QTL refinement using this approach. In collaboration with Dr. Zhao-Bang Zeng (North Carolina State University, Department of Statistics), a probabilistic approach was developed to infer the most likely sire haplotype each son inherited. Now that the most likely sire haplotype has been inferred for each individual in the pedigree, a mixed models approach is
being used to further refine the QTL locations. We hope this method will eventually be incorporated into Windows QTL Cartographer and available to other researchers.
PARTICIPANTS: M.S. Ashwell-PI-planned experiment A.T. O'Nan-technician-helped to train the graduate student A. Alazzeh-PhD student-conducted experiment with help from technician and PI
Impacts
Identification of genes affecting fertility, conformation and body condition score in dairy cattle will allow quantitative geneticists to incorporate genomic information into traditional genetic evaluations. This will allow producers to select superior animals that produce more milk, are healthy, and reproductively sound. This, in turn, will increase global competitiveness and the sustainability of producers and processors in the US. Identification and fine-mapping of QTL affecting these economically important traits is the first step toward gene identification.
Publications
- Alazzeh, A. and Ashwell, M.S. 2007. Fine-mapping of quantitative trait loci on bovine chromosome 15 affecting health and conformation traits in US Holsteins. Plant and Animal Genome XV, San Diego, CA. P545.
|
Progress 10/01/05 to 09/30/06
Outputs
Previous studies have indicated that udder conformation traits are correlated with mastitis incidence in dairy cattle. Previous genome scans identified QTL affecting SCS and several udder conformation traits on BTA15. Each QTL from these scans was placed between 34-55 cM on the genetic map. We were interested in determining if one QTL is responsible for the effects on udder conformation and somatic cell score. Thirteen half-sib families were selected for this study. Eleven markers were genotyped in 923 descendants of the original sire in which the QTL was identified. Regression interval mapping and permutation testing were used to detect across and within family QTL for SCS and 22 conformation traits. Significant and suggested QTL exceeded a chromosome-wise P value of 0.01 and 0.05, respectively were detected. Across family analysis revealed a significant QTL for SCS at 27 cM and thurl width at 96 cM. Further analysis is underway to construct haplotypes and further
refine the location of the QTL. Examination of the bovine genome sequence identifies several genes in QTL region associated with immunity. After further refinement of the QTL locations, SNPs in candidate genes will be evaluated in this complex pedigree to determine if they explain the observed effects on SCS and conformation traits. Continued genetic improvement for milk production has been associated with decreased fertility in US Holstein cattle. A previous study (Ashwell et al., 2004) identified a putative QTL affecting daughter pregnancy rate (DPR) at 54 cM on chromosome 18 in one Holstein grandsire family. We are currently involved in further refinement of the QTL to identify the DNA sequence responsible for the observed effect. Twenty microsatellite markers from BTA18 have been genotyped in 973 animals that were descendants of the original grandsire in which the QTL was identified. Sons of the grandsire as well as 6 grandson and 6 great-grandson families of the original
grandsire were genotyped. In a joint analysis of 13 of the largest families, each containing 10 or more sons, a significant QTL for DPR was detected at 27 cM. QTL affecting DPR was detected in 2 additional sub-families, indicating a putative QTL affecting this trait is most likely segregating within this pedigree. Across-family analysis also detected putative QTL on chromosome 18 affecting productive life at 35 cM, SCS at 33 cM and percent difficult births at 72 cM. The approach used to date has produced very large confidence intervals, preventing further QTL refinement using this approach. In collaboration with Dr. Zhao-Bang Zeng (Department of Statistics), a probabilistic approach is being developed to infer the most likely sire haplotype each son inherited. Once complete, this approach will be used in a mixed models approach to interval mapping. This method will be incorporated into Windows QTL Cartographer and available to researchers. The QTL and/or candidate genes identified in
these projects may be useful for marker-assisted selection to increase the rate of genetic improvement for pregnancy rate, somatic cell score and conformation traits while maintaining genetic improvement for production.
Impacts
Identification of genes affecting fertility, conformation and somatic cell score in dairy cattle will allow quantitative geneticists to incorporate genomic information into traditional genetic evaluations. This will allow producers to select superior animals that produce more milk, are healthy, and reproductively sound. This, in turn, will increase global competitiveness and the sustainability of producers and processors in the US. Identification and fine-mapping of QTL affecting these economically important traits is the first step toward gene identification.
Publications
- Muncie SA, Cassady JP, Ashwell MS. Refinement of quantitative trait loci on bovine chromosome 18 affecting health and reproduction in US Holsteins. Anim. Genet. 2006 37: 273-275.
- Connor EE, Ashwell MS, Schnabel R, Williams JL. Comparative mapping of bovine chromosome 27 with human chromosome 8 near a dairy form QTL in cattle. Cytogenet. Genome Res. 2006 112: 98-102.
|
Progress 10/01/04 to 09/30/05
Outputs
Genetic selection for increased milk production has been associated with decreased fertility in US Holstein cattle. A previous study identified a putative quantitative trait locus (QTL) affecting daughter pregnancy rate on chromosome 18 in one Holstein grandsire family. The goal of this research was to determine the validity of the QTL using additional markers and an extended pedigree. Thirteen microsatellite markers located across chromosome 18 were genotyped in 940 animals that were members of the original sire family in which the QTL was identified. Analysis of the genotypes resulted in detection of the same putative QTL affecting daughter pregnancy rate. A joint analysis of the thirteen largest families, each containing ten or more sons, detected a significant QTL for daughter pregnancy rate. QTL affecting daughter pregnancy rate was detected in two individual sub-families, indicating a putative QTL on chromosome 18 affecting this trait is most likely segregating
within this pedigree. Across-family analysis also detected putative QTL affecting productive life and somatic cell score. Analysis of individual families identified significant and suggestive putative QTL at the chromosome-wise significance level affecting somatic cell score and productive life. A complex pedigree analysis is underway to make full use of the statistical power to refine the QTL region affecting these traits so positional candidate genes can be identified and causative mutations detected. Validation of QTL affecting milk production, fertility, and other economically important traits in dairy cattle has continued using a Canadian Holstein complex pedigree. Approximately twenty microsatellite markers from bovine chromosomes 6, 18, and 27 were genotyped in 783 bulls found in this pedigree. Interval mapping using QTL Express software is being used to analyze the data to determine if QTL appear to be segregating in this Canadian Holstein family. If any chromosomes appear to
contain QTL segregating within this pedigree, more robust analysis methods will be used to harness the full power achieved with the complex pedigree structure. Additional genotyping will be conducted in regions found to contain QTL affecting these economically important traits.
Impacts
Identification of genes affecting fertility, conformation and somatic cell score in dairy cattle will allow quantitative geneticists to incorporate genomic information into traditional genetic evaluations. This will allow producers to select superior animals that produce more milk, are health, and reproductively sound. This, in turn, will increase global competitiveness and the sustainability of producers and processors in the US. Identification and fine-mapping of QTL affecting these economically important traits is the first step toward gene identification.
Publications
- Schnabel, R.D., Sonstegard, T.S., Taylor, J.F. and Ashwell, M.S. (2005) Whole genome scan to detect QTL for milk production, conformation, fertility and functional traits in two U.S. Holstein families. Animal Genetics 36: 408-416.
- Young, J.M., Ashwell, M.S., Rohrer, G.A., Smith, A.L. and Cassady, J.P. (2005) Comparative mapping between the pig (Sus scrofa) and human. Plant and Animal Genome XIII: P557.
- Ashwell, M.S., Heyen, D.W., Weller, J.I., Ron, M., Sonstegard, T.S., Van Tassell, C.P., and Lewin, H.A. (2005) Detection of quantitative trait loci influencing conformation traits and calving ease in Holstein-Friesian cattle. Journal of Dairy Science 88: 4111-4119.
- Boyette, K.E., Ashwell, M.S. and Cassady, J.P. (2005) Characterization of a line of pigs for increased litter size combining the genetic marker and candidate gene approaches. Plant and Animal Genome XIII: P564.
- Muncie, S.A. and Ashwell, M.S. (2005) Fine mapping of chromosome 18 for quantitative trait loci affecting health and reproduction in US Holstein cattle. Plant and Animal Genome XIII: P524.
- Schnabel, R. D., Kim, J.-J., Ashwell, M.S., Sonstegard, T.S., Van Tassell, C.P. Connor, E.E. and Taylor, J.F. (2005) Fine-mapping milk production quantitative trait loci on BTA6: Analysis of the bovine osteopontin gene. Proceedings of the National Academy of Sciences of the USA. 102: 6896-6901.
- Schnabel, R.D., Ashwell, M.S., Van Tassell, C.P., Connor, E.E., Kim, J.-J. and Taylor, J.F. (2005) Fine-mapping milk production QTL on BTA6: analysis of the osteopontin gene. Plant and Animal Genome XIII: P532.
|
Progress 10/01/03 to 09/30/04
Outputs
Microsatellite markers from chromosome 18 were selected for genotyping in a family previously reported to segregate a QTL affecting daughter pregnancy rate. DNA from 984 Holstein bulls was extracted and prepared for high-throughput genotyping methods. Genotyping of ten markers has been completed and data analysis is underway. Identification of QTL affecting body condition score and male/female fertility in a Canadian Holstein family has begun. DNA from 783 bulls was extracted and prepared for genotyping. A step-wise approach is being used for this study, in which markers from chromosomes previously reported to carry QTL affecting these phenotypes have been selected and are currently being genotyped in an attempt to validate putative QTL in this family. Genotypic data generated by Dairy Bull DNA Repository collaborators were analyzed to detect putative QTL affecting calving ease and conformation traits in dairy cattle. Results from this analysis were submitted for
publication and are currently under review. Regions on chromosomes 5, 6, 7, 15, 20, 22, and 26 appear to affect multiple traits, such as conformation, milk production and somatic cell score. Additional research is needed to determine the precise locations of the DNA sequences responsible for these effects. DNA markers flanking these regions may be useful for marker assisted selection programs.
Impacts
Identification of genes affecting fertility, conformation and body condition score in dairy cattle will allow quantitative geneticists to incorporate genomic information into traditional genetic evaluations. This will allow producers to select superior animals that produce more milk, are healthy, and reproductively sound. This, in turn, will increase global competitiveness and the sustainability of producers and processors in the US. Identification and fine-mapping of QTL affecting these economically important traits is the first step toward gene identification.
Publications
- No publications reported this period
|
|