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.

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.

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.

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.

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.

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.

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 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.

The effect of a c.-8G>T polymorphism on the expression of cytochrome b5A and boar taint in pigs.

The level of cytochrome b5A (CYB5A) in pig testis is correlated with boar taint from androstenone and an AF016388:c.-8G>T polymorphism in CYB5A has been linked with low androstenone levels in the fat of pigs. In this study, we developed a polymerase chain reaction-based assay to genotype 1242 boars from eight lines for the c.-8G>T SNP. The c.-8T allele was found in all eight lines at a frequency ranging from 1.8% to 20.3% with an overall frequency of 8.6%. Significant deviations from Hardy-Weinberg equilibrium were found in the Hampshire, Landrace and Yorkshire breeds. The homozygous mutant c.-8TT occurred infrequently and was not found in some lines, but was consistently associated with low androstenone levels in fat. Both CYB5A mRNA and CYB5A protein levels were decreased in the c.-8TT genotype in a subset of Yorkshire boars, suggesting that low levels of CYB5A protein in the c.-8TT mutant were not due to inefficient translation of CYB5A mRNA. There were significant but modest marker effects on fat androstenone levels in Landrace, Yorkshire and a Large White/Duroc cross and fat skatole in Duroc and Sire Line breeds. There was no effect of CYB5A genotype on bulbourethral gland length, suggesting that this SNP will not affect reproductive traits. We conclude that the c.-8G>T SNP in the CYB5A gene has a significant but modest effect on boar taint in male pigs and could be useful in some breeds as part of a panel of SNP markers in a marker-assisted selection programme to produce low boar taint pigs.

Modelling quality variations in commercial Ontario pork production.

This study explores the interactions of sensory and nutritional environment with genotype occurring in current commercial pork production in Ontario, Canada, which may interact to result in poor quality meat. The study focussed on identifying factors and signalling mechanisms that contribute to poor meat quality, in order to develop strategies to reduce the incidence of unacceptable product quality. In the first phase of the work reported here, animal behaviour and muscle metabolism studies were related to meat colour, tenderness and water-holding capacity measurements from commercially-produced pigs killed in a commercial packing plant. A partial least squares analysis was used to determine the most important of the principal production variables, peri-mortem biochemical measures and post-mortem carcass condition variables studied, in terms of their influence on water-holding, toughness and colour (L*-value). Variations between producer and kill day at the slaughterhouse were very strong contributors to variability in these three meat quality parameters, followed by pH variations. A second phase of the study is currently underway to characterize patterns of gene expression related to extremes of end-product quality and to reduce quality variations by nutritional and behavioural management strategies.

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.

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 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.

Effect of testicular steroids on catalytic activities of cytochrome P450 enzymes in porcine liver microsomes.

The testicular steroids androstenone (A), 17beta-oestradiol (E2) and testosterone (T) were tested for their ability to alter CYP2E1 and CYP2A activity in porcine liver microsomes from male and female pigs. This is the first in vitro study indicating that sex steroids have a potential to modify microsomal CYP2E1 activity, the main skatole-metabolising enzyme. A and E2 exerted an inhibitory effect on CYP2E1 mediated hydroxylation of p-nitrophenol to p-nitrocatechol although the mechanism of this inhibition differed for these steroids. The inhibitory effect of A on CYP2E1, as determined by kinetic analysis, might be due to the competitive binding of A and p-nitrophenol to the same site of CYP2E1. Including E2 into the incubations resulted in decreased activities of CYP2E1 in male microsomes through a mixed mode of inhibition. Including pre-incubation steps eliminated this inhibition in male microsomes, and resulted in increased CYP2E1 activities in the microsomes from female pigs. Testosterone was ineffective as an inhibitor of either CYP2E1 or CYP2A activities. Overall, our findings indicate that A and E2 have the potential to modify the catalytic activities of porcine CYP2E1 in vitro. However, the significance of this modification for skatole metabolism in vivo is questionable.

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.

Molecular cloning and regulation of porcine SULT2A1: relationship between SULT2A1 expression and sulfoconjugation of androstenone.

Hydroxysteroid sulfotransferase (SULT2A1) is a key enzyme in the testicular and hepatic metabolism of 5alpha-androstenone, which is a major component of the off-odor and off-flavor in pork known as boar taint. The goals of this study were to determine the role of testicular and hepatic SULT2A1 activity on plasma 5alpha-androstenone sulfate levels, the accumulation of 5alpha-androstenone in adipose tissue, and to gain insight into the regulatory control of SULT2A1. Testicular SULT2A1 activity was negatively correlated (r = -0.57; P < 0.01) with 5alpha-androstenone concentrations in fat. The differences observed in SULT2A1 activity warranted investigation into potential genetic variation within porcine SULT2A1. The cDNA sequence of porcine Sult2A1 was determined to be > 82% homologous to the human, mouse, and rat Sult2A1 genes. A single nucleotide polymorphism was detected within the coding region of the Sult2A1 from individual testes and liver samples; however, this did not affect the amino acid sequence of the enzyme. Western blot analysis determined that animals with high concentrations of 5alpha-androstenone in fat and low SULT2A1 activity had corresponding low levels of SULT2A1 protein compared with animals with low levels of 5alpha-androstenone in fat. Real-time PCR analysis indicated that Sult2A1 mRNA was increased 2.8-fold in animals with high levels of the protein relative to animals with low levels of the protein. Furthermore, we demonstrated the positive role of the nuclear receptors constitutive androstane receptor and pregnane X receptor, as well as the possible role of farnesoid X receptor in the regulation of testicular SULT2A1 activity. Together, the results of this study suggest that differences in SULT2A1 expression can influence 5alpha-androstenone accumulation in fat.

Synthesis of free and sulphoconjugated 16-androstene steroids by the Leydig cells of the mature domestic boar.

This study examined the involvement of sulphoconjugation in the biosynthesis of the 16-androstene steroids in Leydig cells of the mature boar, since the formation of steroid sulphoconjugates can reduce the levels of these steroids that accumulate in fatty tissue. Leydig cells were purified from testes of mature male pigs and incubated with pregnenolone, or various individual 16-androstene steroids for 10 min, 1, 4 and 8h. Sulphoconjugated steroids were recovered by solid-phase extraction followed by solvolysis. Profiles of unconjugated and sulphoconjugated steroids were analysed by HPLC. Steroids present in the sulphoconjugated fractions were purified, derivatised as O-methoxime/trimethylsilyl ethers (MO-TMS), and subsequently identified using gas chromatography-mass spectrometry (GC-MS). The principal metabolite produced from incubations with pregnenolone, androstadienol, androstadienone and 5alpha-androstenone was 3beta-androstenol. 16-Androstene steroids that were sulphoconjugated included 5alpha-androstenone, 3beta-androstenol and 3alpha-androstenol. Approximately 70% of the total amount of each 16-androstene steroid was in its sulphoconjugated form after incubations for 4h or more. The finding that sulphoconjugated 5alpha-androstenone was present in large amounts suggests that this steroid may be converted from a 3-keto to a 3-enol form which is subsequently sulphoconjugated. These findings emphasise the need to consider the impact of sulphoconjugation of the 16-androstene steroids and their role in contributing to boar taint.

Metabolism of the 16-androstene steroids in primary cultured porcine hepatocytes.

The hepatic metabolism of the 16-androstene steroids was investigated using isolated porcine hepatocytes. This study demonstrated that the liver is capable of producing both phase I and phase II steroid metabolites from 16-androstene steroid precursors. 16-Androstene metabolites were recovered by solid-phase extraction and identified by gas chromatography-mass spectrometry (GC-MS). When 5alpha-androstenone was provided as a substrate, both 3beta- and 3alpha-androstenol were produced as well as a metabolite that showed evidence of hydroxylation. Incubations with the various 16-androstene steroids produced metabolic profiles which suggested that the major role of the liver is phase II conjugation. Sulfoconjugated 16-androstene steroids included androstadienol, 5alpha-androstenone, 3beta-, 3alpha-androstenol, and possibly the hydroxylated metabolite of 5alpha-androstenone. It was determined that hydroxysteroid sulfotransferase (HST) is the likely candidate for the sulfoconjugation of the 16-androstene steroids within the liver. Despite the capacity of the hepatocytes to sulfoconjugate the 16-androstene steroids, the principle metabolites produced from incubations with 5alpha-androstenone, 3beta-, and 3alpha-androstenol were glucuronide conjugates, accounting for approximately 68% of all phase II metabolism. These findings underline the importance of steroid conjugation and suggest that hepatic metabolism of the 16-androstene steroids may influence the levels of 5alpha-androstenone present in the circulation, and thus, capable of accumulating in fat.

A comparative evaluation of thiobarbituric acid methods for the determination of malondialdehyde in biological materials.

A comparative evaluation was made of the conventional spectrophotometric procedure and three published high performance liquid chromatographic (HPLC) procedures for the determination of malondialdehyde (MDA) as the thiobarbituric acid (TBA) derivative when applied to liver, fish meal, serum, and urine. Except for urine, spectrophotometric analysis overestimated MDA content. Purification of the TBA-MDA complex obtained from liver and fish meal on reverse phase cartridges was found to entail a loss of complex bound to residual peptides in the trichloracetic acid (TCA) extract. Mincing as opposed to homogenizing liver samples led to a doubling of values for MDA content. Hexanal was a major TBA reactant, in addition to MDA, in all the samples. Acid hydrolysis and heat were necessary for the release of MDA bound to the amino groups of proteins and other amino compounds. Methods for free MDA have limited application to biological materials except short term in vitro preparations such as peroxidizing microsomes, in which free MDA accumulates. On the basis of these and other observations, a modified HPLC procedure for the determination of MDA as the TBA-MDA complex is proposed.