Effects of a coccidiosis challenge on dietary methionine recommendations in broilers.

Broilers are commonly exposed to coccidiosis infections, and the use of dietary strategies to reduce losses in growth performance has practical implications for the poultry industry. Methionine (Met) is typically the first limiting amino acid for broilers and is involved in metabolic and immunological pathways; however, literature is conflicting on how dietary Met requirements are affected by environmental stressors. Our objective was to assess how the Met requirement changes during coccidiosis based on results of growth performance, carcass traits, and health outcomes. Two trials were conducted using 780 male Ross 308 broiler chicks in floor pens randomly assigned to 1 of 12 experimental treatments. All birds received common starter (d 0-10) and finisher (d 24-35, Trial 2 only) diets, and only differed based on their assigned experimental grower diet (d 10-24). Trial 1 experimental grower diets ranged from 2.61 to 6.21 g/kg digestible Met. Trial 2 experimental grower diets were formulated to contain 15% below, at, or 15% above the Met requirement determined in Trial 1. Birds were exposed to a coccidiosis challenge on d 11, with blood and tissue collection (1 bird/pen) on d 18 and carcass processing on d 35 (2 birds/pen) in Trial 2. Data were analyzed using a 1- or 2-way ANOVA. A non-linear regression analysis was conducted in Trial 1 to determine the Met requirement of 4.32 g of digestible Met/kg of diet using BW gain. Coccidiosis infection reduced (P < 0.05) growth performance during the experimental grower and overall study periods in Trial 2. Increasing dietary Met from below requirement to meeting requirement during the grower period improved (P < 0.001) BW gain and feed conversion ratio (FCR), but this effect was only significant between treatments below and above the requirement for the overall study period. There was an interactive effect (P = 0.038) on FCR for the overall study period. These findings provide evidence that the Met requirement is likely increased during coccidiosis based on growth performance outcomes.

Defining optimal dietary starch, oil, and amino acid inclusion levels for broilers experiencing a coccidiosis challenge.

Alternative methods to alleviate coccidiosis in broilers are of interest to producers, including dietary strategies to minimize disruptions in growth rate and efficiency when faced with health challenges. Our objective was to determine optimal combinations of dietary starch, amino acids (AA), and oil to benefit productivity of broilers experiencing Eimeria-induced immune activation. Two trials were conducted using 1,536 male Ross 308 broiler chicks in floor pens randomly assigned to 1 of 17 experimental treatments. All birds received common starter (d 0-10) and finisher (d 24-35) diets, and only differed based on their assigned experimental grower diet (d 10-24). Trial 1 experimental grower diets ranged from 2,700 to 3,300 kcal/kg AME. Trial 2 included 10 experimental grower diets following a simplex lattice design consisting of 3 basal lots formulated to have the highest starch (45.4%), oil (10.2%), or AA density (120, 1.33% digestible Lys) and mixed in 4 equally spaced levels for each component (0, 0.33, 0.67, 1). These mixtures enabled varying densities of AA (80-120% of recommendation), starch:oil (4:1-20:1), and AME (2,940-3,450 kcal/kg). Bird and feeder weights were collected on d 0, 10, 24, and 35, and birds were exposed to an Eimeria challenge on d 11 or 12. In trial 2, excreta samples were collected for AME determination and carcasses were processed on d 36. Data were analyzed using ANOVA, t test, or regression. In Trial 1, BW gain and feed conversion were improved (P < 0.05) by increasing dietary AME. In Trial 2, birds receiving diets containing AA at 93 to 107% of recommendations and higher oil exhibited improved (P < 0.05) performance, but increased starch at the expense of oil reduced performance (P < 0.05). Relative breast and fat pad weights were not influenced by diet in Trial 2. We determined that broilers mildly challenged with Eimeria would exhibit highest BW gain when receiving diets containing 35.8% starch, 8.9% oil, and 101.3% of AA recommendations, which can be utilized by producers to maintain productivity under health-challenged conditions.

Phenobarbital responsiveness conferred by the 5'-flanking region of the rat CYP2B2 gene in transgenic mice.

Phenobarbital (PB) is a prototype for a class of agents that produce marked transcriptional activation of a number of genes, including certain cytochrome P-450s. We used transgenic mouse approaches and multiple gene reporters to assess the functional consequences of specific deletions and site-specific mutations within the 2.5kb 5'-flanking region of the rat CYP2B2 gene. Protein-DNA interactions at the PBRU domain also were characterized. Using the transgenic models, we demonstrate that sequences between -2500 and -1700bp of the CYP2B2 gene are critical for PB induction; mice with 1700 or 800bp of 5'-flanking CYP2B2 sequence are not PB responsive. DNA affinity enrichment techniques and immunoblotting and electromobility shift assays were used to determine that nuclear factor 1 (NF-1) interacts strongly with a site centered at -2200bp in the PB responsive unit (PBRU) of CYP2B2. To test the functional contribution of NF-1 in PB activation, we introduced specific mutations within the PBRU NF-1 element and demonstrated that these mutations completely ablate the binding interaction. However, transgenic mice incorporating the mutant NF-1 sequence within an otherwise wild-type -2500/CYP2B2 transgene maintained full PB responsiveness. These results indicate that, despite the avidity of the respective DNA-protein interaction within the PBRU in vitro, NF-1 interaction is not an essential factor directing PB transcriptional activation in vivo.

Placental permeability for morphine-3-beta-D-glucuronide in the guinea pig.

The fetal Vd and the PS of the major metabolite of morphine, M3G, were studied in pregnant guinea pigs during the last half of gestation. Fetal Vd was determined to be 0.334 ml/g of fetal plus placental weight and did not vary as the gestation progressed. The mean +/- s.e. PS for M3G was 3.7 +/- 0.3 X 10(-5) ml/sec/g and was independent of anaesthesia. This value was consistent with previous studies of other hydrophilic compounds, indicating that diffusion governs the placental passage of M3G. The PS value increased with increasing fetal weight which was consistent with structural changes in the guinea pig placenta as fetal age progresses in late gestation.

Comparison of chronic morphine and placebo infusion in late gestation fetal lambs: effect upon survival and breathing movements.

A placebo-controlled, randomized study of the effect of a 10-day infusion of morphine (MOR) upon fetal survival and fetal breathing movements was done in late gestation lambs. MOR infusion at a rate of 3 mg.hr-1 did not affect fetal survival or the response of fetal breathing movements to hypercapnia. Chronic exposure to MOR increased the height of the integrated diaphragmatic electromyogram signal (IDIA), respiratory drive and inspiratory effort during periods of eucapnia. Respiratory drive was determined by the product of IDIA height and breathing frequency, and inspiratory effort was the quotient of IDIA height divided by inspiratory time. These effects may be related to accumulation of morphine-3-beta-D-glucuronide. Higher doses of MOR, 10 (n = 3) and 30 (n = 1) mg.hr-1, caused seizures and decreased fetal survival.

Phenobarbital induction and tissue-specific expression of the rat CYP2B2 gene in transgenic mice.

To investigate molecular events regulating the transcription of genes inducible by phenobarbital, transgenic mouse strains were developed incorporating rat cytochrome P450 2B2 (CYP2B2) genes. Expression in mouse tissues was analyzed for two series of rat CYP2B2 gene constructs, of 19 and 39 kilobase pairs total length, each containing the entire coding region, introns, and 3'-flanking sequences of CYP2B2, but differing in the respective lengths of 5'-flanking sequence. One group of mice, whose transgene included the complete 2B2 gene but only 800 base pairs of 5'-proximal sequence, were not phenobarbital-inducible in mouse liver or in any extrahepatic tissue; rather, these genes were expressed at very high levels constitutively and selectively in only kidney and liver. A second group of mice with an identical transgene, except for the presence of an additional 19 kilobase pairs of 5'-flanking sequence, expressed 2B2 only in the liver and at high levels only after phenobarbital treatment, analogous to the expression pattern observed for the endogenous CYP2B2 gene in the rat. These results demonstrate that, in vivo, phenobarbital induction and tissue-specific control requires interaction of regulatory elements far upstream of the core CYP2B2 promoter region and upstream of motifs indicated previously as determinants of phenobarbital responsiveness.

Binding of thiopental in neonatal serum.

Protein binding of thiopental was studied in 21 samples of neonatal serum (from placental blood) and compared with protein binding in ten healthy volunteers. These infants ranged between 32 and 43 weeks of gestational age (mean, 37.7 weeks) and the adult age range was from 27 to 54 years (mean, 35.4 years). Because the unbound fraction of the drug is responsible for its pharmacologic effect, a marked difference in the protein binding between neonates and adults may be relevant to the clinician. Blood obtained from freshly delivered placentas or from adult volunteers was allowed to clot and the serum separated from the sample. A portion of the serum was sent for protein and bilirubin analysis and the remainder retained for study. This latter serum was combined with four concentrations of thiopental. These specimens were then ultrafiltered and the amount of thiopental in the ultrafiltrate (unbound) compared with the prefiltered amount (total), as measured by reverse-phase high-performance liquid chromatography. The binding studies were repeated at pH 7.2, 7.4, and 7.6 in both the adult and neonatal serum. Total protein and albumin are significantly less in neonatal serum, whereas bilirubin (total and direct) is significantly higher in neonatal serum than in adult serum (P less than 0.01). Neonatal serum was associated with significantly more unbound thiopental than adult serum at all levels of pH studied (P less than 0.005). Increasing the pH resulted in less free drug in both groups, but this reached statistical significance only in the adult group (P less than 0.025). Drug concentration had no effect on binding in the range examined.(ABSTRACT TRUNCATED AT 250 WORDS)

Elevated retinoic acid receptor beta(4) protein in human breast tumor cells with nuclear and cytoplasmic localization.

The transcription factor retinoic acid receptor beta(2) (RARbeta(2)) is a potent inhibitor of breast cancer cells in vitro, and studies suggest that RARbeta expression is lost in primary breast cancer. Although RARbeta(2) is selectively down-regulated at the mRNA level in breast tumor cells, we show that expression of an RARbeta protein is elevated in five of five breast tumor cell lines relative to normal human mammary epithelial cells. Subsequent analysis identified this protein as the translation product of the human RARbeta(4) transcript. Unlike the previously characterized mouse RARbeta(4) isoform, the human RARbeta(4) retains only half of a DNA-binding domain and lacks a ligand-independent transactivation domain at its N terminus. The RARbeta(4) protein localizes to the cytoplasm and to subnuclear compartments that resemble nuclear bodies. The structure and preliminary characterizations of human RARbeta(4), coupled with the observation that its expression is greatly elevated in breast tumor cell lines, support the hypothesis that RARbeta(4) functions as a dominant-negative repressor of RAR-mediated growth suppression.

Accumulation and clearance of morphine-3-beta-D-glucuronide in fetal lambs.

The time course and extent of morphine-3-beta-D-glucuronide (M3G) production from morphine (MOR) and the clearance of M3G from plasma was studied in the late gestation fetal lamb. MOR was infused at a constant rate into the fetal vena cava and plasma was sampled from the fetus and ewe. Amniotic fluid was also sampled in one animal. M3G was produced in the fetal lamb and accumulated extensively in fetal plasma and amniotic fluid. Seizure activity was observed in two fetal lambs with extremely high plasma concentrations of M3G and MOR. The molar ratio of M3G to MOR in fetal plasma was dependent upon the duration of infusion, reaching a plateau of 60 at about 3 days, but appeared to be independent of the infusion rate over the range studied, 3 to 30 mg hr-1. Maternal plasma MOR and M3G concentrations were substantially less than corresponding fetal plasma concentrations. Using the steady-state plasma concentration of M3G obtained from infusion of MOR and the clearance of M3G derived from single injection of M3G to fetal lambs, the fraction of MOR converted to M3G by the fetus was calculated to be 0.63. Although MOR is conjugated to glucuronic acid in the fetal lamb and excreted into the amniotic fluid it is not readily transferred across the epitheliochorial placenta to the ewe because of restricted permeability.