Genome-wide association study and functional analyses for clinical and subclinical ketosis in Holstein cattle.

Ketosis is one of the most frequent metabolic diseases in high-yielding dairy cows and is characterized by high concentrations of ketone bodies in blood, urine, and milk, causing high economic losses. The search for polymorphic genes, whose alleles have different effects on resistance to developing the disease, is of extreme importance to help select less susceptible animals. The aims of this study were to identify genomic regions associated with clinical and subclinical ketosis (ß-hydroxybutyrate concentration) in North American Holstein dairy cattle and to investigate these regions to identify candidate genes and metabolic pathways associated with these traits. To achieve this, a GWAS was performed for 4 traits: clinical ketosis lactation 1, clinical ketosis lactation 2 to 5, subclinical ketosis lactation 1, and subclinical ketosis lactation 2 to 5. The estimated breeding values from 77,277 cows and 7,704 bulls were deregressed and used as pseudophenotypes in the GWAS. The top-20 genomic regions explaining the largest proportion of the genetic variance were investigated for putative genes associated with the traits through functional analyses. Regions of interest were identified on chromosomes 2, 5, and 6 for clinical ketosis lactation 1; 3, 6, and 7 for clinical ketosis lactation 2 to 5; 1, 2, and 12 for subclinical ketosis lactation 1; and 20, 11, and 25 for subclinical ketosis lactation 2 to 5. The highlighted genes potentially related to clinical and subclinical ketosis included ACAT2 and IGF1. Enrichment analysis of the list of candidate genes for clinical and subclinical ketosis showed molecular functions and biological processes involved in fatty acid metabolism, lipid metabolism, and inflammatory response in dairy cattle. Several genomic regions and SNPs related to susceptibility to ketosis in dairy cattle that were previously described in other studies were confirmed. The novel genomic regions identified in this study aid to characterize the most important genes and pathways that explain the susceptibility to clinical and subclinical ketosis in dairy cattle.

Differential gene expression in dairy cows under negative energy balance and ketosis: A systematic review and meta-analysis.

Development of ketosis in high-producing dairy cows contributes to several animal health issues and highlights the need for a better understanding of the genetic basis of metabolic diseases. To evaluate the pattern of differential gene expression in the liver of cows under negative energy balance (NEB), and under subclinical and clinical ketosis, a meta-analysis of gene expression and genome-wide association studies results was performed. An initial systematic review identified 118 articles based on the key words "cow," "liver," "negative energy balance," "ketosis," "expression," "qPCR," "microarray," "proteomic," "RNA-Seq," and "GWAS." After further screening for only peer-reviewed and pertinent articles for gene expression during NEB and clinical and subclinical ketosis (considering plasma levels of ß-hydroxybutyrate), 20 articles were included in the analysis. From the systematic review, 430 significant SNPs identified by genome-wide association studies (GWAS) were assigned to genes reported in gene expression studies by considering chromosome and base pair positions in the ARS-UCD 1.2 bovine assembly. Venn diagrams were created to integrate the data obtained in the systematic review, and Gene Ontology enrichment analysis was carried out using official gene names. A QTL enrichment analysis was also performed to identify potential positional candidate loci. Twenty-four significant SNPs were located within the coordinates of differentially expressed genes located on chromosomes 2, 3, 6, 9, 11, 14, 27, and 29. Three significant metabolic pathways were associated with NEB and subclinical and clinical ketosis. In addition, 2 important genes, PPARA (peroxisome proliferator activated receptor alpha) and ACACA (acetyl-coenzyme A carboxylase α), were identified, which were differentially expressed in the 3 metabolic conditions. The PPARA gene is involved in the regulation of lipid metabolism and fatty liver disease and the ACACA gene encodes an enzyme that catalyzes the carboxylation of acetyl-coenzyme A to malonyl-coenzyme A, which is a rate-limiting step in fatty acid synthesis. Gene network analysis revealed co-expression interactions among 34 genes associated with functions involving fatty acid transport and fatty acid metabolism. For the annotated QTL, 9 QTL were identified for ketosis. The genes FN1 (fibronectin 1) and PTK2 (protein tyrosine kinase 2), which are mainly involved in cell adhesion and formation of extracellular matrix constituents, were enriched for QTL previously associated with the trait "ketosis" on chromosome 2 and for the trait "milk iron content" on chromosome 14, respectively. This integration of gene expression and GWAS data provides an additional understanding of the genetic background of NEB and subclinical and clinical ketosis in dairy cattle. Thus, it is a useful approach to identify biological mechanisms underlying these metabolic conditions in dairy cattle.

The characterization of androstenone transport in boar plasma.

The transport of steroids by plasma proteins influences the amount of steroid available for uptake by the target tissue. In the boar, androstenone is transported to the adipose tissue where it accumulates to cause an off-odour or off-flavour in pork, known as boar taint. The mechanism of the transport of androstenone in the boar remains unclear, and the plasma protein responsible for binding androstenone has yet to be identified. Therefore, the purpose of the present study was to characterize the binding of androstenone to plasma proteins in the boar. The binding specificity of androstenone to plasma proteins was first investigated using a HPLC gel filtration method. [3H]-androstenone was incubated with plasma in the presence or absence of unlabeled competitors and the displacement of androstenone from plasma proteins was measured. In the presence of excess unlabeled competitors, [3H]-androstenone was only partially displaced from plasma proteins, indicating it binds to a low affinity high capacity plasma protein. Binding kinetics studies were also conducted to characterize the binding of androstenone and dehydroepiandrosterone (DHEA) to plasma proteins. The Bmax of androstenone and DHEA was approximately the same (89.1% and 92.3%, respectively). However, the binding affinity (K) of androstenone was 6.5 fold greater than DHEA (0.39 nmol/ml and 0.06 nmol/ml, respectively). Affinity chromatography was used to remove albumin from the plasma proteins. Following incubations with androstenone and DHEA, the binding observed in the albumin free protein fraction was reduced 2.6 and 2.1 fold, respectively relative to the binding in the albumin protein fractions. These results provide direct evidence that androstenone is transported non-specifically by albumin in the plasma of the boar.

Candidate gene association analyses for ketosis resistance in Holsteins.

High-yielding dairy cattle are susceptible to ketosis, a metabolic disease that negatively affects the health, fertility, and milk production of the cow. Interest in breeding for more robust dairy cattle with improved resistance to disease is global; however, genetic evaluations for ketosis would benefit from the additional information provided by genetic markers. Candidate genes that are proposed to have a biological role in the pathogenesis of ketosis were investigated in silico and a custom panel of 998 putative single nucleotide polymorphism (SNP) markers was developed. The objective of this study was to test the associations of these new markers with deregressed estimated breeding values (EBV) for ketosis. A sample of 653 Canadian Holstein cows that had been previously genotyped with a medium-density SNP chip were regenotyped with the custom panel. The EBV for ketosis in first and later lactations were obtained for each animal and deregressed for use as pseudo-phenotypes for association analyses. Results of the mixed inheritance model for single SNP association analyses suggested 15 markers in 6 unique candidate genes were associated with the studied trait. Genes encoding proteins involved in metabolic processes, including the synthesis and degradation of fatty acids and ketone bodies, gluconeogenesis, lipid mobilization, and the citric acid cycle, were identified to contain SNP associated with ketosis resistance. This work confirmed the presence of previously described quantitative trait loci for dairy cattle, suggested novel markers for ketosis-resistance, and provided insight into the underlying biology of this disease.

Effects of fatty acid glyceride product SILOhealth 104 on the growth performance and carcass composition of broiler chickens.

Butyric acid is the primary energy source for colonocytes, and has shown potential as an alternative to in-feed antibiotics, due to its antimicrobial activity and positive effects on production performance traits of broiler chickens. SILOhealth 104 (SILO S.P.A., Florence, Italy) is a commercial product mainly containing mono- and di-glycerides of butyrate with a small portion of propionic, caprylic, capric, and lauric acid mono- and di-glycerides. Its effects on broiler performance and carcass composition have yet to be evaluated. Four-hundred-eighty day-old male Ross 308 birds were divided into different dietary treatment groups with equal starting weights and fed a diet containing 0, 500, 1,000, 2,000, or 3,000 ppm of SILOhealth 104 for 35 days. There were no significant differences in overall average daily gain or feed: gain ratio with the addition of SILOhealth 104 to the diets (P > 0.05). At 5 wk of age, abdominal fat weight was reduced in birds supplemented with SILOhealth 104 in a dose-responsive manner (P < 0.05), while breast muscle weight increased with supplementation, with significant increases in 2,000 ppm and 3,000 ppm birds compared to controls (P < 0.05). A significant reduction in gene expression of both forkhead box protein O4 and myostatin, 2 factors that can inhibit protein synthesis, was found in the breast muscle of all SILOhealth 104 treated birds (P < 0.05). In addition, gene expression in the adipose tissue, including acetyl-CoA carboxylase alpha and lipoprotein lipase, which are associated with lipid metabolism, was significantly decreased and increased, respectively, by the supplementation of SILOhealth 104 (P < 0.05). These data suggest that the components of SILOhealth 104 can positively affect the deposition of muscle, while reducing abdominal fat deposition in broiler chickens.

Age at castration (surgical or immunological) impacts carcass characteristics and meat quality of male pigs.

In order to accurately estimate body composition at slaughter and to meet specific market targets, the influence of age at time of castration (surgical or immunological) on body composition and boar taint indicators must be determined for male pigs. In all, 48 males were randomly assigned to one of four management regimens: (1) entire male pigs (EM), (2) EM surgically castrated at ~40 kg BW and 10 weeks of age (late castrates; LC), (3) conventional, early surgical castrates (within 4 days of birth; EC) and (4) EM immunized with a gonadotropin-releasing hormone (GnRH) analog (primary dose at 30 kg BW and 8 weeks of age; booster dose at 70 kg and 14 weeks of age; IM). Pigs were fed corn and soybean meal-based diets that were not limiting in essential nutrients. Back fat was sampled on days -3, 8, 18 and 42, relative to administering the booster dose of GnRH analog at day 0, to determine androstenone concentrations (n=8 or 9/group). Fat androstenone concentrations in IM were lower than EM between days 8 and 42 after administering the booster dose (173 v. 863 ng/g, respectively; P<0.01), and were not different from surgically castrated males (EC and LC) after day 18. Slaughter occurred at ~115 kg BW, 42 days (6 weeks) after administering the booster dose for IM, and 10 and 20 weeks after surgical castration for LC and EC, respectively (n=8 or 9/group). At slaughter, live BW, liver weight as a percent of live BW, dissectible bone as a percent of cold carcass side, body protein and water contents and whole-body protein deposition decreased with time after surgical castration (linear; P<0.05), whereas dressing percentage, dissectible fat, probe fat depth and body fat content increased with time after surgical castration (linear; P<0.05). The IM had intermediate dressing percentage and dissected fat to EM and EC, whereas liver weight as a percent of live BW and body protein and lipid contents were not different from EM. Whole-body lipid deposition tended to be greater in IM than in EM between 14 and 20 weeks of age (373 v. 286 g/d; P=0.051). In conclusion, castration of male pigs after 6 weeks of age has a lasting effect on physical and chemical body composition. The relationship between time after castration and body composition may be developed to predict carcass composition and can be used to determine the ideal immunization schedule aimed at specific markets in the future.

Effects of supplementation level and feeding schedule of butyrate glycerides on the growth performance and carcass composition of broiler chickens.

Mixed mono- and tributyrate glycerides have been used for effective delivery of butyrate to the gut to benefit broilers. However, limited information is available on the efficacy of butyrate glycerides individually and in combination with different levels and feeding schedules. The present study has first investigated the effects of monobutyrin at inclusion levels of zero, 500, 1,000, 2,000, and 3,000 ppm on the performance of broilers, and second, the effects of its combination with tributyrin. In the monobutyrin trial, there were no overall significant differences in average daily gain or feed efficiency. However, 2,000 ppm birds had significantly decreased abdominal fat deposition compared to controls (P ≤ 0.05), and the breast muscle deposition increased in a dose-response manner to the supplementation of monobutyrin (P ≤ 0.05). The combination trial tested 5 treatment groups: control, 500 ppm tributyrin + 500 ppm monobutyrin (5T5M), 500 ppm tributyrin + 500 ppm monobutyrin staggered (5T5Ms), 500 ppm tryibutyrin + 2,000 ppm monobutyrin (5T20M), or 500 ppm tributyrin + 2,000 ppm monobutyrin staggered (5T20Ms). In staggered groups, birds were fed tributyrin for one wk followed by 2 wk of monobutyrin, after which the feed was butyrate glyceride free. The non-staggered groups had constant inclusions levels through the 5 weeks. There were no significant differences in average daily gain or feed efficiency among groups. At 5 wk of age, all treatment groups except for 5T5Ms had significantly lower relative abdominal fat weight compared to control birds (P ≤ 0.05), although 5T5Ms birds demonstrated a trend for a decrease (P = 0.095). Relative breast muscle weight was significantly increased only in 5T5M birds over control birds at 5 wk of age (P ≤ 0.05). Serum biochemistry revealed significant changes in factors relating to muscle growth and fat deposition (P ≤ 0.05). These results indicate a consistent shift in lipid metabolism with the addition of butyrate glycerides and that the deposition of breast muscle may be highest with the incorporation of butyrate glycerides at a moderate level for the duration of development.

Dynamics of nitrogen retention in entire male pigs immunized against gonadotropin-releasing hormone.

Immunization against GnRH reduces the occurrence of boar-taint-causing compounds in entire male pigs and is likely to alter growth performance, including whole-body protein deposition (PD; N retention × 6.25). Thirty-six male pigs were used to determine the effects of immunization against GnRH with GnRH analog on N retention and plasma urea nitrogen (PUN; a measure of AA catabolism). Four treatments were used: 1) conventional, early castrates (EC), 2) entire male pigs (EM), 3) entire male pigs immunized with GnRH analog (IM), and 4) entire male pigs surgically castrated after 6 wk of age (between 25 and 40 kg BW; late castrates; LC). The GnRH analog was injected at 30 and 70 kg BW. Within each of 9 litters, 4 males were randomly assigned to the 4 treatments. Pigs were fed corn- and soybean-meal-based diets that were not limiting in essential nutrients for high PD. Five consecutive N balances were conducted between d -9 and -4, 1 and 7, 9 and 16, 20 and 26, and 30 and 36, relative to administration of the booster dose of GnRH analog at d 0. Blood was sampled on d -4, -1, 2, 5, 8, 11, 14, 19, 28, and 37. There was an interactive effect of treatment and time on N retention (P < 0.001). Across periods, N retention for EC and LC were similar (32.7 vs. 33.6 g/d) and lower than EM (39.2 g/d, P < 0.001). The N retention in EM and IM were similar up to d 7 (37.8 vs. 38.5 g/d), tended to be greater for EM than IM between d 9 and 16 (38.4 vs. 34.9 g/d, P = 0.07) and was greater for EM than IM after d 20 (40.9 vs. 34.9 g/d, P < 0.05). Between d 9 and 36, N retention in IM was similar to EC and LC. The PUN concentrations were similar in EC and LC across sampling times (15.50 vs. 15.86 mg/dL) and greater than EM (9.33 mg/dL, P < 0.05). The PUN concentrations were similar in EM and IM up to d 5 (9.88 vs. 9.59 mg/dL), tended to be less in EM than IM on d 8 (9.08 vs. 11.85 mg/dL, P < 0.10), and were lower in EM than IM from d 11 to 37 (8.94 vs. 14.80 mg/dL, P < 0.05). After d 8, PUN concentrations were similar for IM, EC, and LC (14.31, 15.13, and 15.55 mg/dL, respectively). In conclusion, the results of the current study show that N retention and PUN patterns in EC and LC are very similar and lower than those in EM. Between d 7 and 16 after administration of the booster dose of GnRH analog, N retention and PUN in IM changed gradually from EM levels to approach levels in EC and LC, which should be considered when developing feeding programs for IM.

Identification of single nucleotide polymorphisms in genes involved in digestive and metabolic processes associated with feed efficiency and performance traits in beef cattle.

Discovery of genetic mutations that have a significant association with economically important traits would benefit beef cattle breeders. Objectives were to identify with an in silico approach new SNP in 8 genes involved in digestive function and metabolic processes and to examine the associations between the identified SNP and feed efficiency and performance traits. The association between SNP and daily DMI, ADG, midpoint metabolic weight (MMWT), residual feed intake (RFI), and feed conversion ratio (FCR; the ratio of average daily DMI to ADG) was tested in discovery and validation populations using a univariate mixed-inheritance animal model fitted in ASReml. Substitution effect of the T allele of SNP rs41256901 in protease, serine, 2 (trypsin 2; PRSS2) was associated with FCR (-0.293 ± 0.08 kg DMI kg(-1) BW gain; P < 0.001) and RFI (-0.199 ± 0.08 kg; P < 0.01) and although not significant in the validation population, the phase of association remained. In the cholecystokinin B receptor (CCKBR) gene, genotypes in rs42670351 were associated with RFI (P < 0.05) whereas genotypes in rs42670352 were associated with RFI (P = 0.002) and DMI (P < 0.05). Substitution of the G allele in rs42670352 was associated with DMI (-0.236 ± 0.12 kg; P = 0.055) and RFI (-0.175 ± 0.09 kg; P = 0.05). Substitution of the G allele of SNP rs42670353 was associated with ADG (0.043 ± 0.02 kg/d; P < 0.01) and FCR (0.114 ± 0.05 kg BW gain kg(-1) DMI; P < 0.05). In the validation dataset, SNP rs42670352 in gene CCKBR was significant for RFI and DMI and had the same phase of associations; SNP rs42670353 was significantly associated with FCR with same phase of association and the C allele in SNP rs42670351 was validated as decreasing DMI, RFI, and FCR. Substituting the G allele of SNP rs42670352 in CCKBR2 was associated with decreasing DMI and RFI in the validation study. New SNP were reported in genes PRSS2 and CCKBR, being associated with feed efficiency and performance traits in beef cattle. The association between these SNP with fertility, carcass, and meat quality traits must still be tested.

The roles of different porcine cytochrome P450 enzymes and cytochrome b5A in skatole metabolism.

Boar taint is the unfavourable odour and taste from pork fat, which results in part from the accumulation of skatole (3-methylindole, 3MI). The key enzymes in skatole metabolism are thought to be cytochrome P450 2E1 (CYP2E1) and cytochrome 2A (CYP2A); however, the cytochrome P450 (CYP450) isoform responsible for the production of the metabolite 6-hydroxy-3-methylindole (6-OH-3MI, 6-hydroxyskatole), which is thought to be involved in the clearance of skatole, has not been established conclusively. The aim of this study was to characterize the role of porcine CYP450s in skatole metabolism by expressing them individually in the human embryonic kidney HEK293-FT cell line. This system eliminates the problems of the lack of specificity of antibodies, inhibitors and substrates for CYP450 isoforms in the pig, and contributions of any other CYP450s that would be present. The results show that pig CYP1A1, CYP2A19, CYP2C33v4, CYP2C49, CYP2E1 and CYP3A and human CYP2E1 (hCYP2E1) are all capable of producing the major skatole metabolite 3-methyloxyindole (3MOI), as well as indole-3-carbinol (I3C), 5-hydroxy-3-methylindole (5-OH-3MI), 6-OH-3MI, 2-aminoacetophenone (2AAP) and 3-hydroxy-3-methyloxindole. CYP2A19 produced the highest amount of the physiologically important metabolite 6-OH-3MI, followed by porcine CYP2E1 and CYP2C49; CYP2A19 also produced more 6-OH-3MI than hCYP2E1. Co-transfection with CYB5A increased the production of skatole metabolites by some of the CYP450s, suggesting that CYB5A plays an important role in the metabolism of skatole. We also show the utility of this expression system to check the specificity of selected substrates and antibodies for porcine CYP450s. Further information regarding the abundance of different CYP450 isoforms is required to fully understand their contribution to skatole metabolism in vivo in the pig.

Single nucleotide polymorphisms in collagenous lectins and other innate immune genes in pigs with common infectious diseases.

Innate immune recognition of pathogens involves various surface receptors and soluble proteins that precede agglutination, complement activation, phagocytosis, and the adaptive immune response. Mannan-binding lectins (MBLs), ficolins (FCNs) and surfactant protein A (SP-A) are soluble collagenous lectins that bind surface structures of various bacteria, viruses and fungi. Some single nucleotide polymorphisms (SNPs) in collagenous lectin genes of humans and other species, including pigs, have been implicated in variation in susceptibility to infectious and inflammatory diseases. In this study we determined the frequencies of 13 SNP alleles of MBL-A, MBL-C, ficolin-α, ficolin-ß, and SP-A in 1324 healthy pigs and 461 pigs diagnosed with common infectious diseases at necropsy. For comparison, we also analyzed 12 other SNP alleles in several other innate immune genes, including galectins and TLRs. Several SNPs within genes encoding porcine MBL-A, MBL-C and SP-A were more frequent in pigs diagnosed at necropsy with various diseases or pathogens. These findings suggest that several collagenous lectin SNPs are associated with disease susceptibility and therefore might be genetic markers of impaired innate immune function.

Efficacy of non-nutritive sorbent materials as intestinal-binding agents for the control of boar taint.

In many countries, male pigs are castrated to prevent boar taint, but this practice raises concerns about animal welfare and reduces the production efficiency of pork. The objective of this study was to develop dietary manipulations to prevent boar taint. We evaluated the effectiveness of adding activated carbon (AC) or Tween-60 (Tween; polyoxyethylene sorbitan monostearate) to pig finishing diets to reduce levels of androstenone (AND) and skatole in plasma and fat of entire male pigs. Boars (159 ± 2 days of age at the start of the experiment) were fed diets supplemented with either 5% AC or 5% Tween for 28 days followed by either 14 or 28 days of recovery. Plasma samples were collected at experimental days 0, 7, 14, 21, 28, 42 and 56, and backfat biopsies were taken at experimental days 0, 28, 42 and 56. Feeding AC significantly (P < 0.05) reduced the levels of AND in plasma by day 28 compared to day 0 and by day 42 in fat compared to day 0. AC treatment also decreased levels of oestrone sulphate (E(1)S) in plasma by day 7 compared to day 0. Treatment with Tween significantly decreased (P < 0.05) the levels of plasma AND by day 28 from levels at day 0. Tween treatment did not significantly affect levels of fat AND or plasma E(1)S compared to day 0; however, fat AND levels decreased between days 28 and 42 following treatment with Tween (P < 0.05). Levels of plasma E(1)S, plasma AND and fat AND for control boars remained constant throughout the experiment. Skatole plasma concentrations were very low and did not vary significantly (P > 0.05) from day 0 for any treatment, but fat skatole levels decreased by day 42 in the Tween treatment group. Importantly, there was no difference in growth rate between the control and experimental groups. We conclude that adding AC or Tween to finishing diets for boars can reduce the levels of plasma and fat AND, but further work is needed to confirm the effects of these treatments on reducing fat skatole levels.

In vitro assessment of the effectiveness of non-nutritive sorbent materials as binding agents for boar taint compounds.

Boar taint, an off-odor and an off-flavor in the meat from some uncastrated male pigs, is due to high levels of the testicular steroid hormone, androstenone, and the indole, skatole. Thus far, there are no known methods for controlling both androstenone and skatole through dietary means. We tested the adsorbent agents, cholestyramine (CH), activated carbon (AC), tween-60 (Tween), bentonite (BNT) and polyvinylpolypyrrolidone (PVPP) for binding androstenone, estrone (E(1)), estrone sulfate (E(1)S) and skatole from buffer solutions in an in vitro system. The goal was to determine the potential utility of these binding agents as feed additives to control boar taint. Michaelis-Menten analysis was utilized to determine the effectiveness of the adsorbents. At pH 7.4, E(1)S was bound to AC and CH with the highest B(max) (maximum binding), whereas Tween and AC had the greatest B(max) for E(1). The B(max) for skatole at pH 7.4 was highest for AC, CH and PVPP. AC had a higher B(max) for androstenone than CH and Tween. The B(max) values at pH 3.0 with E(1)S for AC and CH were essentially 100%, whereas the binding of Tween to E(1)S at pH 3.0 decreased by 49.5% from binding at pH 7.4 (P < 0.05). The Ad(int) values, which represent efficiency of binding, illustrated that AC bound E(1), androstenone and skatole with greater efficiency than the other binding agents at pH 7.4, whereas AC bound E(1)S as efficiently as CH. We conclude that AC was the most effective adsorbent agent for binding E(1), E(1)S, androstenone and skatole in vitro, followed by CH, Tween, PVPP and lastly BNT. These adsorbent agents may be useful for binding boar taint compounds in in vivo studies to decrease the risk of boar taint.

Chlorzoxazone metabolism by porcine cytochrome P450 enzymes and the effect of cytochrome b5.

Chlorzoxazone (CLZ) is a commonly used nontoxic in vivo and in vitro probe for the assessment of CYP2E1 activity. Human CYP1A1 and CYP3A4 have also been shown to contribute to CLZ metabolism. For pigs to be a potential model system for humans, it is necessary that human and pig cytochromes P450 (P450) have similar metabolizing capabilities. Therefore, CLZ metabolizing capabilities and specificities of porcine P450s were investigated. In this study, the complete coding regions of six porcine P450s were amplified from liver cDNA and cloned into pcDNA3.1/V5-His TOPO vector. Expression vectors for the individual P450s and microsomal cytochrome b(5) (CYB5A) were expressed in the human embryonic kidney HEK-293FT cell line to investigate their role in CLZ metabolism. As with the human enzymes, porcine CYP2E1 (K(m) = 290.3 microM and V(max) = 4980 pmol/h/mg total protein) and CYP1A1 (K(m) = 159.5 microM and V(max) = 1650 pmol/h/mg total protein) both contribute to CLZ metabolism. In addition, porcine CYP2A19 and CYP2C33v4 also metabolize the substrate, with K(m) = 212.1 microM and V(max) = 6680 pmol/h/mg total protein and K(m) = 126.3 microM and V(max) = 2100 pmol/h/mg total protein, respectively, whereas CYP3A does not. CYB5A augmented CYP2E1 and CYP2C33v4 activity in the pig, with a significant increase in activity of 85 and 73% compared with control, respectively. Thus, CLZ should be used with caution as a probe for CYP2E1 activity in the pig. However, further information regarding the abundance of different P450 isoforms is needed to fully understand their contribution in microsomal, hepatocyte, and in vivo systems in the pig.

Characterization of the porcine constitutive androstane receptor (CAR) and its splice variants.

Porcine constitutive androstane receptor (CAR; NR1I3) was cloned and compared for homology and activity with mouse and human CAR (mCAR, hCAR). Porcine CAR (pgCAR) was 86% and 75% homologous to hCAR at the nucleotide and protein levels. Five alternatively spliced variants of pgCAR were identified, each of which generated a truncated protein product. Real-time polymerase chain reaction (PCR) analyses showed that these variants were present in pig liver cDNA samples from 4.61% to 9.20% of total pgCAR. pgCAR and hCAR responded similarly to more ligands than did hCAR and mCAR. The known hCAR agonist (6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde-O-(3,4-dichlorobenzyl)oxime (CITCO) activated pgCAR, while the murine agonist 1,4 bis[2-(3,5-dichloropyridyloxy)] benzene (TCPOBOP) had no effect. 5beta-dihydrotestosterone was identified as a novel inverse agonist of both pgCAR and hCAR. pgCAR splice variant 2 (SV2) had a dose-dependent dominant negative effect on the activity of wild-type pgCAR in dual luciferase assays. SV2 had no effect against pgPXR (pregnane X receptor) or pgFXR (farnesoid X receptor) activity when using PXR- or FXR-specific reporters.

Effect of dietary omega-3 and omega-6 fatty acids on clotting activities of Factor V, VII and X in fatty liver haemorrhagic syndrome-susceptible laying hens.

1. The relationship between concentrations of omega-3 and omega-6 fatty acids in plasma and Factor V, VII and X clotting activities was determined using a crossover feeding trial with diets supplemented with either soy oil or flax oil. 2. Laying hens on the soy diet, which is high in omega-6 fatty acids, had substantially higher clotting activity for all three factors compared to laying hens on the flax diet that was high in omega-3 fatty acids. 3. Positive associations were seen between liver haemorrhage score and the percentage of liver weight and between the percentage of liver weight and the severity of haemorrhagic and fatty changes seen on histology. 4. These results support the hypothesis that concentrations of omega-6 and omega-3 fatty acids in plasma affect clotting activity; however, there was no relationship between the extent of liver haemorrhages and the composition of plasma fatty acids.

The impact of meloxicam on postsurgical stress associated with cautery dehorning.

The objectives were to determine the duration of the stress response associated with cautery dehorning and to assess the effectiveness of the nonsteroidal anti-inflammatory drug meloxicam (Metacam, 20 mg/mL solution for injection) for reducing that response. Sixty Holstein heifer calves were blocked by age and randomly assigned to receive an i.m. injection of meloxicam or a placebo (0.5 mg/kg). All calves were given a lidocaine cornual nerve block delivered 5 mL per side 10 min before dehorning. To establish baseline values, calves were sham dehorned 24 h before actual dehorning. Blood samples were taken via indwelling jugular catheters at 0, 0.5, 1, 1.5, 2, 4, 6, and 24 h after the procedure. Heart and respiratory rates were also taken at these times. Data were analyzed using PROC MIXED in SAS. Analysis of covariance was employed to assess the difference between sham and dehorning at each time period. Dehorning was associated with elevated serum cortisol (d -1: 33.9 +/- 1.26; d 0: 46.2 +/- 2.33 nmol/L) and heart rate (d -1: 108 +/- 1.8; d 0: 109.4 +/- 2.4 beats per minute) in both groups for 24 h, and elevated respiratory rate (sham: 42.2 +/- 1.95 vs. dehorning: 45.1 +/- 2.19 respirations per minute) in both groups for 6 h. A treatment x time interaction was found for cortisol, with meloxicam calves having lower serum cortisol than controls until 6 h after dehorning (meloxicam: 49.7 +/- 4.37 vs. control: 63.0 +/- 6.94 nmol/L). There was no difference between the treatment groups at 24 h (meloxicam: 35.2 +/- 2.74 and control: 34.8 +/- 3.64 nmol/L of cortisol). Overall, the changes in heart rates (increase meloxicam: 3.74 +/- 0.96 vs. control: 4.70 +/- 1.87) and respiratory rates (increase meloxicam: 2 +/- 0.1 vs. control: 4 +/- 0.2) were greater in the control group compared with the meloxicam group. These results indicate that meloxicam reduced the physiological stress response to dehorning.

The role of porcine cytochrome b5A and cytochrome b5B in the regulation of cytochrome P45017A1 activities.

Male pigs are routinely castrated to prevent the accumulation of testicular 16-androstene steroids, in particular 5alpha-androst-16-en-3-one (5alpha-androstenone), which contribute to an off-odour and off-flavour known as boar taint. Cytochrome P450C17 (CYP17A1) catalyses the key regulatory step in the formation of the 16-androstene steroids from pregnenolone by the andien-beta synthase reaction or the synthesis of the glucocorticoid and sex steroids via 17alpha-hydroxylase and C17,20 lyase pathways respectively. We have expressed CYP17A1, along with cytochrome P450 reductase (POR), cytochrome b5 reductase (CYB5R3) and cytochrome b5 (CYB5) in HEK-293FT cells to investigate the importance of the two forms of porcine CYB5, CYB5A and CYB5B, in both the andien-beta synthase as well as the 17alpha-hydroxylase and C17,20 lyase reactions. Increasing the ratio of CYB5A to CYP17A1 caused a decrease in 17alpha-hydroxylase (p<0.013), a transient increase in C17,20 lyase, and an increase in andien-beta synthase activity (p<0.0001). Increasing the ratio of CYB5B to CYP17A1 also decreased 17alpha-hydroxylase, but did not affect the andien-beta synthase activity; however, the C17,20 lyase, was significantly increased. These results demonstrate the differential effects of two forms of CYB5 on the three activities of porcine CYP17A1 and show that CYB5B does not stimulate the andien-beta synthase activity of CYP17A1.

Biochemical, nutritional and genetic effects on boar taint in entire male pigs.

Pork odour is to a great extent affected by the presence of malodorous compounds, mainly androstenone and skatole. The present review outlines the current state of knowledge about factors involved in the regulation of androstenone and skatole in entire male pigs. Androstenone is a pheromonal steroid synthesised in the testes and metabolised in the liver. Part of androstenone accumulates in adipose tissue causing a urine-like odour. Skatole is produced in the large intestine by bacterial degradation of tryptophan and metabolised by hepatic cytochrome P450 enzymes and sulphotransferase. The un-metabolised part accumulates in adipose tissue, causing faecal-like odour. Androstenone levels are mostly determined by genetic factors and stage of puberty, whereas skatole levels in addition to genetic background and hormonal status of the pigs are also controlled by nutritional and environmental factors. To reduce the risk of tainted carcasses entering the market, male pigs are surgically castrated in many countries. However, entire males compared to castrates have superior production characteristics: higher growth rate, better feed efficiency and leaner carcasses. Additionally, animal welfare aspects are currently of particular importance in light of increasing consumers' concerns. Nutrition, hormonal status, genetic influence on boar taint compounds and the methods to develop genetic markers are discussed. Boar taint due to high levels of skatole and androstenone is moderately heritable and not all market weight entire males have boar taint; it should thus be possible to select for pigs that do not have boar taint. In these studies, it is critical to assess the steroidogenic potential of the pigs in order to separate late-maturing pigs from those with a low genetic potential for boar taint. A number of candidate genes for boar taint have been identified and work is continuing to develop genetic markers for low boar taint. More research is needed to clarify the factors involved in the development of boar taint and to develop additional methods to prevent the accumulation of high concentrations of skatole and androstenone in fat. This review proposes those areas requiring further research.

Influence of plasma lipid composition on activity of Factors V, VII and X in Single Comb White Leghorn and Fatty Liver Haemorrhagic Syndrome-susceptible laying hens.

1. Plasma lipids were investigated to determine whether they influence the biological activities of specific coagulation proteins Factors V, VII and X. 2. Factor activities decreased when lipids were depleted from the plasma of Single Comb White Leghorn (SCWL) and Fatty Liver Haemorrhagic Syndrome-susceptible (FLHS) laying hens. 3. Addition of lipids removed from SCWL laying hens and FLHS-susceptible laying hens into lipid-depleted plasma of both bird strains caused an increase and decrease, respectively, in Factors V and X activities. 4. Omega-3 fatty acids were negatively correlated to Factors V, VII and X activities. When bird strain was considered, it was significant for Factor X in SCWL laying hens. Omega-6 fatty acids were positively correlated with Factors VII and X for FLHS-susceptible laying hens. 5. The results suggest that the type of fatty acid in plasma phospholipids influences the activities of Factors V, VII and X and by altering lipid composition in the plasma, activities of coagulation factors may be affected.