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.

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.

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.

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.

Characterizing the variability in transcript levels across breeds and sex in swine for genes that play a role in drug metabolism may shed light on breed and sex differences in drug metabolism. The objective of the study is to determine if there is heterogeneity between swine breeds and sex in transcript levels for genes previously shown to play a role in drug metabolism for animals administered flunixin meglumine or fenbendazole. Crossbred nursery female and castrated male pigs (n = 169) spread across 5 groups were utilized. Sires (n = 15) of the pigs were purebred Duroc, Landrace, Yorkshire or Hampshire boars mated to a common sow population. Animals were randomly placed into the following treatments: no drug (control), flunixin meglumine, or fenbendazole. One hour after the second dosing, animals were sacrificed and liver samples collected. Quantitative Real-Time PCR was used to measure liver gene expression of the following genes: SULT1A1, ABCB1, CYP1A2, CYP2E1, CYP3A22 and CYP3A29. The control animals were used to investigate baseline transcript level differences across breed and sex. Post drug administration transcript differences across breed and sex were investigated by comparing animals administered the drug to the controls. Contrasts to determine fold change were constructed from a model that included fixed and random effects within each drug. Significant (P-value <0.007) basal transcript differences were found across breeds for SULT1A1, CYP3A29 and CYP3A22. Across drugs, significant (P-value <0.0038) transcript differences existed between animals given a drug and controls across breeds and sex for ABCB1, PS and CYP1A2. Significant (P <0.0038) transcript differences across breeds were found for CYP2E1 and SULT1A1 for flunixin meglumine and fenbendazole, respectively. The current analysis found transcript level differences across swine breeds and sex for multiple genes, which provides greater insight into the relationship between flunixin meglumine and fenbendazole and known drug metabolizing genes.