M. S. Ashwell

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.

Identification of stable normalization genes for quantitative real-time PCR in porcine articular cartilage

BackgroundExpression 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. The purpose of this study was to identify the most stable housekeeping genes in porcine articular cartilage subjected to a mechanical injury from a panel of 10 candidate genes.ResultsTen candidate housekeeping genes were evaluated in three different treatment groups of mechanically impacted porcine articular cartilage. The genes evaluated were: beta actin, beta-2-microglobulin, glyceraldehyde-3-phosphate dehydrogenase, hydroxymethylbilane synthase, hypoxanthine phosphoribosyl transferase, peptidylprolyl isomerase A (cyclophilin A), ribosomal protein L4, succinate dehydrogenase flavoprotein subunit A, TATA box binding protein, and tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein—zeta polypeptide. The stability of the genes was measured using geNorm, BestKeeper, and NormFinder software. The four most stable genes measured via geNorm were (most to least stable) succinate dehydrogenase flavoprotein, subunit A, peptidylprolyl isomerase A, glyceraldehyde-3-phosphate dehydrogenase, beta actin; the four most stable genes measured via BestKeeper were glyceraldehyde-3-phosphate dehydrogenase, peptidylprolyl isomerase A, beta actin, succinate dehydrogenase flavoprotein, subunit A; and the four most stable genes measured via NormFinder were peptidylprolyl isomerase A, succinate dehydrogenase flavoprotein, subunit A, glyceraldehyde-3-phosphate dehydrogenase, beta actin.ConclusionsBestKeeper, geNorm, and NormFinder all generated similar results for the most stable genes in porcine articular cartilage. The use of these appropriate reference genes will facilitate accurate gene expression studies of porcine articular cartilage and suggest appropriate housekeeping genes for articular cartilage studies in other species.

The addition of high manganese to a copper-deficient diet further depresses copper status and growth of cattle

A study was conducted evaluating the effect of long-term Cu deficiency, with or without high Mn, on growth, gene expression and Cu status of beef cattle. Twenty-one Angus calves were born to cows receiving one of the following treatments: (1) 10 mg supplemental Cu/kg DM (+Cu); (2) no supplemental Cu and 2 mg Mo/kg DM ( - Cu); (3) - Cu diet plus 500 mg supplemental Mn/kg DM ( - Cu+Mn). Calves were weaned at approximately 183 d of age and individually fed throughout the growing and finishing phases. Plasma Cu was lower (P < 0.01) in - Cu calves compared with +Cu calves while high dietary Mn further depressed (P < 0.01) plasma Cu in - Cu+Mn calves v. - Cu calves. Liver Cu concentrations in +Cu calves were greater (P < 0.01) than in - Cu calves, with no differences between - Cu and - Cu+Mn calves. The daily body-weight gain of +Cu calves was greater (P < 0.01) than - Cu calves during the period from birth to weaning, but did not differ during the growing phase. - Cu+Mn calves gained less (P < 0.05) than - Cu calves during the growing phase. DM intake was lower (P < 0.01) in - Cu+Mn calves v. - Cu calves, and did not differ among +Cu and - Cu calves. The relative gene expression of cytochrome c oxidase in the liver was lower (P < 0.05) in - Cu calves compared with +Cu or - Cu+Mn calves. In conclusion, feeding a Cu - deficient diet in combination with high Mn negatively affected the growth and Cu status of beef cattle.

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 1 2

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.

Trans-10, cis-12-conjugated linoleic acid alters hepatic gene expression in a polygenic obese line of mice displaying hepatic lipidosis.

The trans-10, cis-12 isomer of conjugated linoleic acid (CLA) causes a rapid reduction of body and adipose mass in mice. In addition to changes in adipose tissue, numerous studies have reported alterations in hepatic lipid metabolism. Livers of CLA-fed mice gain mass, partly due to lipid accumulation; however, the precise molecular mechanisms are unknown. To elucidate these mechanisms, we examined fatty acid composition and gene expression profiles of livers from a polygenic obese line of mice fed 1% trans-10, cis-12-CLA for 14 days. Analysis of gene expression data led to the identification of 1393 genes differentially expressed in the liver of CLA-fed male mice at a nominal P value of .01, and 775 were considered significant using a false discovery rate (FDR) threshold of .05. While surprisingly few genes in lipid metabolism were impacted, pathway analysis found that protein kinase A (PKA) and cyclic adenosine monophosphate (cAMP) pathways signaling pathways were affected by CLA treatment and 98 of the 775 genes were found to be regulated by hepatocyte nuclear factor 4alpha, a transcription factor important in controlling liver metabolic status.

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.

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.

A genome‐wide association study of direct gestation length in US Holstein and Italian Brown populations

Direct gestation length influences economically important traits in dairy cattle that are related to birth and peri-natal survival of the calf. The objective of this study was to identify single nucleotide polymorphisms (SNPs) that are significantly associated with direct gestation length through a genome-wide association study. Data used in the analysis included 7,308,194 cow gestation lengths from daughters of 4743 United States Holstein sires in the Cooperative Dairy DNA Repository population and 580,157 gestation lengths from 749 sires in the Italian Brown population. Association analysis included 36,768 and 35,082 SNPs spanning all autosomes for Holstein and Brown Swiss, respectively. Multiple shrinkage Bayesian was employed. Estimates of heritability for both populations were moderate, with values of 0.32 (±0.03) and 0.29 (±0.02) for Holstein and Brown Swiss, respectively. A panel of SNPs was identified, which included SNPs that have significant effects on direct gestation length, of which the strongest candidate region is located on chromosome 18. Two regions not previously linked to direct calving ease and calf survival were identified on chromosome 7 and 28, corresponding to regions that contain genes related to embryonic development and foetal development. SNPs were also identified in regions that have been previously mapped for calving difficulty and longevity. This study identifies target regions for the investigation of direct foetal effects, which are a significant factor in determining the ease of calving.

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.

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.