Environmental assessment of three egg production systems--Part I: Monitoring system and indoor air quality.

To comprehensively assess conventional vs. some alternative laying-hen housing systems under U.S. production conditions, a multi-institute and multi-disciplinary project, known as the Coalition for Sustainable Egg Supply (CSES) study, was carried out at a commercial egg production farm in the Midwestern United States over two single-cycle production flocks. The housing systems studied include a conventional cage house (200,000 hen capacity), an aviary house (50,000 hen capacity), and an enriched colony house (50,000 hen capacity). As an integral part of the CSES project, continual environmental monitoring over a 27-month period described in this paper quantifies indoor gaseous and particulate matter concentrations, thermal environment, and building ventilation rate of each house. Results showed that similar indoor thermal environments in all three houses were maintained through ventilation management and environmental control. Gaseous and particulate matter concentrations of the enriched colony house were comparable with those of the conventional cage house. In comparison, the aviary house had poorer indoor air quality, especially in wintertime, resulting from the presence of floor litter (higher ammonia levels) and hens' activities (higher particulate matter levels) in it. Specifically, daily mean indoor ammonia concentrations had the 95% confidence interval values of 3.8 to 4.2 (overall mean of 4.0) ppm for the conventional cage house; 6.2 to 7.2 (overall mean of 6.7) ppm for the aviary house; and 2.7 to 3.0 (overall mean of 2.8) ppm for the enriched colony house. The 95% confidence interval (overall mean) values of daily mean indoor carbon dioxide concentrations were 1997 to 2170 (2083) ppm for the conventional cage house, 2367 to 2582 (2475) ppm for the aviary house, and 2124 to 2309 (2216) ppm for the enriched colony house. Daily mean indoor methane concentrations were similar for all three houses, with 95% confidence interval values of 11.1 to 11.9 (overall mean of 11.5) ppm. The 95% confidence interval values (overall mean) of daily mean PM10 and PM2.5 concentrations, in mg/m3, were, respectively, 0.57 to 0.61 (0.59) and 0.033 to 0.037 (0.035) for the conventional cage house, 3.61 to 4.29 (3.95) and 0.374 to 0.446 (0.410) for the aviary house, and 0.42 to 0.46 (0.44) and 0.054 to 0.059 (0.056) for the enriched colony house. Investigation of mitigation practices to improve indoor air quality of the litter-floor aviary housing system is warranted.

Impact of commercial housing systems and nutrient and energy intake on laying hen performance and egg quality parameters.

The US egg industry is exploring alternative housing systems for laying hens. However, limited published research related to cage-free aviary systems and enriched colony cages exists related to production, egg quality, and hen nutrition. The laying hen's nutritional requirements and resulting productivity are well established with the conventional cage system, but diminutive research is available in regards to alternative housing systems. The restrictions exist with limited availability of alternative housing systems in research settings and the considerable expense for increased bird numbers in a replicate due to alternative housing system design. Therefore, the objective of the current study was to evaluate the impact of nutrient and energy intake on production and egg quality parameters from laying hens housed at a commercial facility. Lohmann LSL laying hens were housed in three systems: enriched colony cage, cage-free aviary, and conventional cage at a single commercial facility. Daily production records were collected along with dietary changes during 15 production periods (28-d each). Eggs were analyzed for shell strength, shell thickness, Haugh unit, vitelline membrane properties, and egg solids each period. An analysis of covariance (ANCOVA) coupled with a principal components analysis (PCA) approach was utilized to assess the impact of nutritional changes on production parameters and monitored egg quality factors. The traits of hen-day production and mortality had a response only in the PCA 2 direction. This finds that as house temperature and Met intake increases, there is an inflection point at which hen-day egg production is negatively effected. Dietary changes more directly influenced shell parameters, vitelline membrane parameters, and egg total solids as opposed to laying hen housing system. Therefore, further research needs to be conducted in controlled research settings on laying hen nutrient and energy intake in the alternative housing systems and resulting impact on egg quality measures.

Comparative evaluation of three egg production systems: Housing characteristics and management practices.

This paper is an integral part of the special publication series that arose from the multidisciplinary and multi-institutional project of the Coalition for Sustainable Egg Supply (CSES). The CSES project involves 3 housing systems for egg production at the same research farm site in the Midwest, USA, namely, a conventional cage (CC) house, an aviary (AV) house, and an enriched colony (EC) house. The CC house (141.4 m L×26.6 m W×6.1 m H) had a nominal capacity of 200,000 hens (6 hens in a cage at a stocking density of 516 cm2/hen), and the cages were arranged in 10 rows, 8 tiers per cage row, with a perforated aisle walkway at 4-tier height. The AV house (154.2 m L×21.3 m W×3.0 m H) and the EC house (154.2 m L×13.7 m W×4.0 m H) each had a nominal capacity of 50,000 hens. The AV house had 6 rows of aviary colonies, and the EC house had 5 rows of 4-tier enriched colonies containing perches, nestbox, and scratch pads (60 hens per colony at a stocking density of 752 cm2/hen). The overarching goal of the CSES project, as stated in the opening article of this series, was to comprehensively evaluate the 3 egg production systems from the standpoints of animal behavior and well-being, environmental impact, egg safety and quality, food affordability, and worker health. So that all the area-specific papers would not have to repeat a detailed description of the production systems and the management practices, this paper is written to provide such a description and to be used as a common reference for the companion papers.

Environmental assessment of three egg production systems--Part II. Ammonia, greenhouse gas, and particulate matter emissions.

As an integral part of the Coalition for Sustainable Egg Supply (CSES) Project, this study simultaneously monitored air emissions of 3 commercially operated egg production systems at the house level and associated manure storage over 2 single-cycle flocks (18 to 78 wk of age). The 3 housing systems were 1) a conventional cage house (CC) with a 200,000-hen capacity (6 hens in a cage at a stocking density of 516 cm2/hen), 2) an enriched colony house (EC) with a 50,000-hen capacity (60 hens per colony at a stocking density of 752 cm2/hen), and 3) an aviary house (AV) with a 50,000-hen capacity (at a stocking density of 1253 to 1257 cm2/hen). The 3 hen houses were located on the same farm and were populated with Lohmann white hens of the same age. Indoor environment and house-level gaseous (ammonia [NH3] and greenhouse gasses [GHG], including carbon dioxide [CO2], methane [CH4], and nitrous oxide [N2O]) and particulate matter (PM10, PM2.5) emissions were monitored continually. Gaseous emissions from the respective manure storage of each housing system were also monitored. Emission rates (ERs) are expressed as emission quantities per hen, per animal unit (AU, 500 kg live BW), and per kilogram of egg output. House-level NH3 ER (g/hen/d) of EC (0.054) was significantly lower than that of CC (0.082) or AV (0.112) (P<0.05). The house-level CO2 ER (g/hen/d) was lower for CC (68.3) than for EC and AV (74.4 and 74.0, respectively), and the CH4 ER (g/hen/d) was similar for all 3 houses (0.07 to 0.08). The house-level PM ER (mg/hen/d), essentially representing the farm-level PM ER, was significantly higher for AV (PM10 100.3 and PM2.5 8.8) than for CC (PM10 15.7 and PM2.5 0.9) or EC (PM10 15.6 and PM2.5 1.7) (P<0.05). The farm-level (house plus manure storage) NH3 ER (g/hen/d) was significantly lower for EC (0.16) than for CC (0.29) or AV (0.30) (P<0.05). As expected, the magnitudes of GHG emissions were rather small for all 3 production systems. Data from this study enable comparative assessment of conventional vs. alternative hen housing systems regarding air emissions and enhance the U.S. national air emissions inventory for farm animal operations.

Site-specific prodrug activation by antibody-beta-lactamase conjugates: regression and long-term growth inhibition of human colon carcinoma xenograft models.

Antibody-directed catalysis (ADC) is a two-step method for the delivery of chemotherapeutic agents in which enzyme-antibody conjugate, prelocalized to antigen-bearing tumor cells, catalyzes the site-specific conversion of prodrug to drug. An ADC system consisting of F(ab')-beta-lactamase conjugates and a cephalosporin derivative of the oncolytic agent 4-desacetylvinblastine-3-carboxhydrazide was investigated. The ability of the system to mediate antitumor activity was compared with that of free drug given alone and with covalent drug-antibody conjugates in LS174T and T380 colon carcinoma xenografts in nude mice. Efficacy increased from moderate tumor growth inhibition by using free 4-desacetylvinblastine-3-carboxhydrazide to tumor regression and long-term stabilization with the ADC system. Labile covalent drug-antibody conjugates prepared from the same antibodies were less effective than ADC and required much higher antibody doses. The antigens KS1/4, carcinoembryonic antigen, and tumor-associated glycoprotein-72, TAG-72, present on the model cell lines, were chosen to investigate the effect of differences in subcellular location and expression heterogeneity on the efficacy of ADC delivery. Response was equivalent with the three tumor antigens. Hence, heterogeneous expression and membrane shedding of carcinoembryonic antigen and TAG-72, did not diminish the suitability of these antigens as targets for ADC therapy. In contrast, drug-antibody conjugate efficacy was more sensitive to subcellular location and heterogeneity. Thus, ADC is a highly effective form of immunochemotherapy in preclinical models, with applicability toward a variety of antigen targets.

Assessment of lighting needs by W-36 laying hens via preference test.

Light intensity, spectrum and pattern may affect laying hen behaviors and production performance. However, requirements of these lighting parameters from the hens' standpoint are not fully understood. This study was conducted to investigate hens' needs for light intensity and circadian rhythm using a light tunnel with five identical compartments each at a different fluorescent light intensity of <1, 5, 15, 30 or 100 lux. The hens were able to move freely among the respective compartments. A group of four W-36 laying hens (23 to 30 weeks of age) were tested each time, and six groups or replicates were conducted. Behaviors of the hens were continuously recorded, yielding data on daily time spent, daily feed intake, daily feeding time, and eggs laid under each light intensity and daily inter-compartment movement. The results show that the hens generally spent more time in lower light intensities. Specifically, the hens spent 6.4 h (45.4%) at 5 lux, 3.0 h (22.1%) at 15 lux, 3.1 h (22.2%) at 30 lux and 1.5 h (10.3%) at 100 lux under light condition; and an accumulation of 10.0 h in darkness (<1 lux) per day. The 10-h dark period was distributed intermittently throughout the day, averaging 25.0±0.4 min per hour. This hourly light-dark rhythm differs from the typical commercial practice of providing continuous dark period for certain part of the day (e.g. 8 h at night). Distributions of daily feed intake (87.3 g/hen) among the different light conditions mirrored the trend of time spent in the respective light intensity, that is, highest at 5 lux (28.4 g/hen, 32.5% daily total) and lowest at 100 lux (5.8 g/hen, 6.7%). Hen-day egg production rate was 96.0%. Most of the eggs were laid in <1 lux (61.9% of total) which was significantly higher than under other light intensities (P<0.05). Findings from this study offer insights into preference of fluorescent light intensity by the laying hens. Further studies to assess or verify welfare and performance responses of the hens to the preferred lighting conditions and rhythm over extended periods are recommended.

Synthesis and evaluation of peptidyl Michael acceptors that inactivate human rhinovirus 3C protease and inhibit virus replication.

Human rhinovirus, the chief cause of the common cold, contains a positive-sense strand of RNA which is translated into a large polyprotein in infected cells. Cleavage of the latter to produce the mature viral proteins required for replication is catalyzed in large part by a virally encoded cysteine proteinase (3Cpro) which is highly selective for -Q approximately GP- cleavage sites. We synthesized peptidyl derivatives of vinylogous glutamine or methionine sulfone esters (e.g., Boc-Val-Leu-Phe-vGln-OR: R = Me, 1; R = Et, 2) and evaluated them as inhibitors of HRV-14 3C protease (3Cpro). Compounds 1 and 2 and several related tetra- and pentapeptide analogues rapidly inactivated 3Cpro with submicromolar IC50 values. Electrospray mass spectrometry confirmed the expected 1:1 stoichiometry of 3Cpro inactivation by 1, 2, and several other analogues. Compound 2 also proved to be useful for active site titration of 3Cpro, which has not been possible heretofore because of the lack of a suitable reagent. In contrast to 1, 2, and congeners, peptidyl Michael acceptors lacking a P4 residue have greatly reduced or negligible activity against 3Cpro, consistent with previously established structure-activity relationships for 3Cpro substrates. Hydrolysis of the P1 vinylogous glutamine ester to a carboxylic acid also decreased inhibitory activity considerably, consistent with the decreased reactivity of acrylic acids vs acrylic esters as Michael acceptors. Incorporating a vinylogous methionine sulfone ester in place of the corresponding glutamine derivative in 1 also reduced activity substantially. Compounds 1 and 2 and several of their analogues inhibited HRV replication in cell culture by 50% at low micromolar concentrations while showing little or no evidence of cytotoxicity at 10-fold higher concentrations. Peptidyl Michael acceptors and their analogues may prove useful as therapeutic agents for pathologies involving cysteine proteinase enzymes.

Synthesis and biological activity of the putative metabolites of the atypical antipsychotic agent tiospirone.

Putative oxidative metabolites of the lead antipsychotic agent tiospirone (1) were synthesized to assist in the identification of the authentic metabolic products found in human urine samples. Thus far, six authentic metabolites have been correlated to the synthetic species. The putative metabolites were further examined in vitro to assess their central nervous system therapeutic potential. SAR analysis of these derivatives indicates that hydroxyl substitution, particularly in the azaspirodecanedione region of the molecule, diminishes the dopamine D-2 affinity of the species without significantly altering the serotonin type-1A and type-2 interactions. In addition, an increase in alpha 1-adrenergic affinity appears to be linked to the attenuation of effects at the dopamine receptors. The biological profile of the 6-hydroxytiospirone metabolite 42 was exemplary in these respects and the in vivo actions of this compound suggest potent antipsychotic potential with a minimal liability for extrapyramidal side effects (EPS). While compound 42 has been unambiguously characterized as an actual human metabolite of tiospirone, the role of 42 in the observed antipsychotic activity of the parent drug, if any, has not yet been determined.

Potent human immunodeficiency virus type 1 protease inhibitors that utilize noncoded D-amino acids as P2/P3 ligands.

Noncoded D-amino acids have been designed to replace the quinaldic amide-asparaginyl moiety (P2/P3 ligand) found in several potent human immunodeficiency virus (HIV) protease inhibitors such as LY289612. The substituted nitrogen, optimally an N-methanesulfonyl moiety, served as a CH2CONH2 (asparagine side chain mimic), while the amino acid side chain became the backbone and P3 ligand of these novel inhibitors. Compounds derived from S-aryl-D-cysteine proved to be potent HIV protease inhibitors which also exhibited potent whole cell antiviral activity. Oxidation of the cysteines to the sulfoxide or sulfone oxidation states resulted in significant improvements in potency. For example, the compound derived from N-(methyl-sulfonyl)-2-S-naphthylcysteine sulfone, 17c, was a 3.5 nM inhibitor of HIV protease which inhibited the spread of virus in MT4 cells with an IC50 = 4.3 nM. Compounds 17c,g,i were found to be orally bioavailable in a rat model.

Development of analytical and preparative chromatographic separations of novel growth hormone secretagogue compounds.

Chromatographic separations of new growth hormone secretagogue compounds were developed to support structure-activity relationship (SAR) studies in conjunction with lead optimization. These new compounds differed from Merck's MK-677 by having two chiral centers and thus diastereomeric mixtures were generated. Separation of initial compounds in the SAR was achieved on a Kromasil C18 column using an ammonium acetate buffer and acetonitrile. However, additional candidates were not separable on C18 columns and a chiral Kromasil CHI-DMB column was used to resolve the diastereomeric compounds. The Kromasil CHI-DMB packing was also used in a preparative chromatographic system to resolve multigram quantities of secretagogue candidates for testing. Chiral separations of different intermediates were also developed in support of evolution of an asymmetric synthetic route. This report summarizes development of the preparative chromatographic system used to purify diastereomeric mixtures and chiral separations of intermediates in the synthesis.

A Physiologic Profile Comparing Levels and Styles of Female Dancers.

In brief: A physiologic profile comparing 39 female dancers was undertaken to better understand the factors distinguishing four levels and styles of dance: professional ballet (PB), professional modern, university ballet, and university modern. The PB dancers had significantly lower VO2 max values on the tread-mill as well as lower peak blood lactate levels following the Wingate test for anaerobic capacity than the other dancers. The PB dancers also had a significantly higher isokinetic hamstring-quadriceps ratio than the modern dancers. These findings suggest that physiologic capacity may differ among female dancers according to their levels and styles of dance.

Development of in vitro peptide substrates for human rhinovirus-14 2A protease.

Purified 2A protease from human rhinovirus serotype-14 (HRV14) was unable to efficiently cleave a 16-mer peptide representing its authentic cis-cleavage site on the viral polyprotein, implying that in vivo cis cleavage by this enzyme might be very different from its in vitro trans activity. Presence of a serine at position P2 and a leucine at P2' in the 16-mer peptide was found to be responsible for the low peptide cleavage efficiency. To search for an efficient peptide substrate for HRV14 2A, small peptides derived from other rhinovirus 2A protease cleavage sites were synthesized and tested. These results suggested that the N-terminal 8 amino acids were sufficient for HRV14 2A cleavage to occur, although the P1' and P2' residue identities were important to the cleavage of peptides with amino acids occupying both sides of the scissile bond. On the basis of the 2A substrate requirements, a sensitive fluorometric assay for the viral 2A proteases was developed using peptides with anthranilide and 3-nitrotyrosine as the resonance energy transfer donor/quencher pair. Our data indicated that these fluorescent peptide substrates were suitable for 2A protease characterization and inhibitor evaluation.

Preparation and characterization of a beta-lactamase-Fab' conjugate for the site-specific activation of oncolytic agents.

Antibody-directed catalysis (ADC) is a two-step method for the targeted delivery of chemotherapeutic agents in which enzyme-antibody conjugates, prelocalized to antigen-bearing cells, activate prodrugs designed to be substrates for the enzyme. An enzyme-Fab' conjugate exhibiting both native beta-lactamase activity and immunoreactivity toward carcinoembryonic antigen (CEA) was constructed. Treatment of CEA-expressing LS174T cells with this conjugate imparted beta-lactamase activity to the cells; beta-lactamase activity was not imparted by treatment with unconjugated beta-lactamase and not to CEA negative cells treated with conjugate. Cephalosporin-based prodrugs, and other substrates synthesized as model compounds, were found to have wide variations in their kinetic parameters toward the conjugate, with kcat values ranging from 16 to 3300 s-1 and KM values ranging from 5 to 160 microM. The prodrug derived from desacetylvinblastine-3-carboxylic acid hydrazide (DAVLBHYD) was studied in vitro and found to be 5-fold less cytotoxic to LS174T cells than the parent DAVLBHYD. For antigen-positive cells preincubated with conjugate, however, the prodrug showed the same potency as the parent drug. Thus, the combination of conjugate and prodrug appears to provide antigen-dependent toxicity to tumor cells.

Site-specific prodrug activation by antibody-beta-lactamase conjugates: preclinical investigation of the efficacy and toxicity of doxorubicin delivered by antibody directed catalysis.

Antibody directed catalysis (ADC), the catalytic conversion of prodrugs to drugs by enzymes localized at disease targets by appropriate monoclonal antibodies, has shown promise in the treatment of cancer in nude mouse xenograft models. We investigated this concept using antibody enzyme conjugates constructed from beta-lactamase and Fab's reactive with carcinoembryonic antigen, CEA, and tumor associated glycoprotein, TAG-72, to convert prodrugs that are cephalosporin sulfoxide derivatives into oncolytic drugs. Previous work focused on ADC delivery of the potent vinca alkaloid derivative desacetylvinblastine carboxhydrazide (DAVLBHYD). In the current study the ability of the system to deliver doxorubicin was tested in MCF7 breast carcinoma xenografts and OVCAR3 ovarian carcinoma xenografts, and in T380 and LS174T colon tumor xenografts for comparison with previous DAVLBHYD results. ADC enhanced the delivery of doxorubicin in the model systems investigated. Tumor growth suppression was equivalent to or greater than that observed with free doxorubicin at its maximum tolerated dose (MTD). In contrast to the DAVLBHYD results, ADC delivery of doxorubicin did not regress tumors, but did result in a substantial increase in the MTD.