Genetic relationship between purebred and crossbred sow longevity.

BACKGROUND: The overall breeding objective for a nucleus swine selection program is to improve crossbred commercial performance. Most genetic improvement programs are based on an assumed high degree of positive relationship between purebred performance in a nucleus herd and their relatives' crossbred performance in a commercial herd. The objective of this study was to examine the relationship between purebred and crossbred sow longevity performance. Sow longevity was defined as a binary trait with a success occurring if a sow remained in the herd for a certain number of parities and including the cumulative number born alive as a measure of reproductive success. Heritabilities, genetic correlations, and phenotypic correlations were estimated using THRGIBBS1F90. RESULTS: Results indicated little to no genetic correlations between crossbred and purebred reproductive traits. This indicates that selection for longevity or lifetime performance at the nucleus level may not result in improved longevity and lifetime performance at the crossbred level. Early parity performance was highly correlated with lifetime performance indicating that an indicator trait at an early parity could be used to predict lifetime performance. This would allow a sow to have her own record for the selection trait before she has been removed from the herd. CONCLUSIONS: Results from this study aid in quantifying the relationship between purebred and crossbred performance and provide information for genetic companies to consider when developing a selection program where the objective is to improve crossbred sow performance. Utilizing crossbred records in a selection program would be the best way to improve crossbred sow productivity.

Feed efficiency effects on barrow and gilt behavioral reactivity to novel stimuli tests.

Increasing feed efficiency is an important goal for improving sustainable pork production and profitability for producers. To study feed efficiency, genetic selection based on residual feed intake (RFI) was used to create 2 divergent lines. Low-RFI pigs consume less feed for equal weight gain compared to their less efficient, high-RFI counterparts. Therefore, our objective was to assess how a pig's behavioral reactivity toward fear-eliciting stimuli related to RFI selection and improvement of feed efficiency. In this study, behavioral reactivity of pigs divergently selected for RFI was evaluated using human approach (HAT) and novel object (NOT) tests. Forty low-RFI and 40 high-RFI barrows and gilts ( = 20 for each genetic line; 101 ± 9 d old) from ninth-generation Yorkshire RFI selection lines were randomly selected and evaluated once using HAT and once using NOT over a 2-wk period utilizing a crossover experimental design. Each pig was individually tested within a 4.9 × 2.4 m test arena for 10 min; behavior was evaluated using live and video observations. The test arena floor was divided into 4 zones; zone 1 being oral, nasal, and/or facial contact with the human (HAT) or orange traffic cone (NOT) and zone 4 being furthest from the human or cone and included the point where the pig entered the arena. During both HAT and NOT, low-RFI pigs entered zone 1 less frequently compared to high-RFI pigs ( ≤ 0.03). During NOT, low-RFI pigs changed head orientation more frequently ( = 0.001) but attempted to escape less frequently (low-RFI = 0.97 ± 0.21 vs. high-RFI = 2.08 ± 0.38; = 0.0002) and spent 2% less time attempting to escape compared to high-RFI pigs ( = 0.04). Different barrow and gilt responses were observed during HAT and NOT. During HAT, barrows spent 2% more time within zone 1 ( = 0.03), crossed fewer zone lines ( < 0.0001), changed head orientation less frequently ( = 0.002), and froze less frequently compared to gilts ( = 0.02). However, during NOT, barrows froze more frequently ( = 0.0007) and spent 2% longer freezing ( = 0.05). When the behavior and RFI relationship was examined using odds ratios, decreasing RFI by 1 kg/d decreased the odds of freezing by 4 times but increased the odds of attempting to escape by 5.26 times during NOT ( ≤ 0.04). These results suggest that divergent selection for RFI resulted in subtle behavioral reactivity differences and did not impact swine welfare with respect to responses to fear-eliciting stimuli.

Measuring the efficacy of flunixin meglumine and meloxicam for lame sows using a GAITFour pressure mat and an embedded microcomputer-based force plate system.

Pain associated with lameness on farm is a negative affective state and has a detrimental impact on individual farm animal welfare. Animal pain can be managed utilizing husbandry tools and through pharmacological approaches. Nonsteroidal anti-inflammatory drugs including meloxicam and flunixin meglumine are compounds used in many species for pain management because they are easy to administer, long lasting, and cost-effective. Assessing an animal's biomechanical parameters using such tools as the embedded microcomputer-based force plate system and GAITFour pressure mat gait analysis walkway system provides an objective, sensitive, and precise means to detect animals in lame states. The objectives of this study were to determine the efficacy of meloxicam and flunixin meglumine for pain mitigation in lame sows using the embedded microcomputer-based force plate system and GAITFour pressure mat gait analysis walkway system. Lameness was induced in 24 mature mixed-parity sows using a chemical synovitis model and compared 3 treatments: meloxicam (1.0 mg/kg per os), flunixin meglumine (2.2 mg/kg intramuscular) and sterile saline (intramuscular). Weight distribution (kg) for each foot was collected twice per second for a total of 5 min for each time point using the embedded microcomputer-based force plate system. Stride time, stride length, maximum pressure, activated sensors, and stance time were collected using 3 quality walks (readings) for each time point using the GAITFour pressure mat gait analysis walkway system. Sows administered flunixin meglumine or meloxicam tolerated more weight on their lame leg compared with saline sows (P < 0.005). Sows administered flunixin meglumine or meloxicam had smaller differences in stance time, maximum pressure, and activated sensors between the sound and lame legs compared with saline-treated sows between 37 and 60 h after lameness induction (P < 0.03). In conclusion, flunixin meglumine and meloxicam administration mitigated pain sensitivity in sows after lameness induction when pain sensitivity was evaluated with the embedded microcomputer-based force plate system and GAITFour pressure mat gait analysis walkway system. Analgesic drugs may be a key tool to manage negative pain affective states associated with lameness.

Using classification trees to detect induced sow lameness with a transient model.

Feet and legs issues are some of the main causes for sow removal in the US swine industry. More timely lameness detection among breeding herd females will allow better treatment decisions and outcomes. Producers will be able to treat lame females before the problem becomes too severe and cull females while they still have salvage value. The objective of this study was to compare the predictive abilities and accuracies of weight distribution and gait measures relative to each other and to a visual lameness detection method when detecting induced lameness among multiparous sows. Developing an objective lameness diagnosis algorithm will benefit animals, producers and scientists in timely and effective identification of lame individuals as well as aid producers in their efforts to decrease herd lameness by selecting animals that are less prone to become lame. In the early stages of lameness, weight distribution and gait are impacted. Lameness was chemically induced for a short time period in 24 multiparous sows and their weight distribution and walking gait were measured in the days following lameness induction. A linear mixed model was used to determine differences between measurements collected from day to day. Using a classification tree analysis, it was determined that the mean weight being placed on each leg was the most predictive measurement when determining whether the leg was sound or lame. The classification tree's predictive ability decreased as the number of days post-lameness induction increased. The weight distribution measurements had a greater predictive ability compared with the gait measurements. The error rates associated with the weight distribution trees were 29.2% and 31.3% at 6 days post-lameness induction for front and rear injected feet, respectively. For the gait classification trees, the error rates were 60.9% and 29.8% at 6 days post-lameness induction for front and rear injected feet, respectively. More timely lameness detection can improve sow lifetime productivity as well as animal welfare.

Evaluation of mechanical and thermal nociception as objective tools to measure painful and nonpainful lameness phases in multiparous sows.

The objective of this study was to quantify pain sensitivity differences using mechanical nociception threshold (MNT) and thermal nociception threshold (TNT) tests when sows were in painful and nonpainful transient lameness phases. A total of 24 mixed parity crossbred sows (220.15 ± 21.23 kg) were utilized for the MNT test, and a total of 12 sows (211.41 ± 20.21 kg) were utilized for the TNT test. On induction day (D0), all sows were anesthetized and injected with Amphotericin B (10mg/mL) in the distal interphalangeal joint space in both claws of one randomly selected hind limb to induce transient lameness. Three days were compared: (1) D-1 (sound phase, defined as 1 d before induction), (2) D+1 (most lame phase, defined as 1 d after induction), and (3) D+6 (resolution phase, defined as 6 d after induction). After completion of the first round, sows were given a 7-d rest period and then the procedures were repeated with lameness induced in the contralateral hind limb. During the MNT test, pressure was applied perpendicularly to 3 landmarks in a randomized sequence for each sow: 1) middle of cannon on the hind limb (cannon), 2) 1 cm above the coronary band on the medial hind claw (medial claw), and 3) 1 cm above the coronary band on the lateral hind claw (lateral claw). During the TNT test, a radiant heat stimulus was directed 1 cm above the coronary band. The data were analyzed using the MIXED procedure in SAS with sow as the experimental unit. Differences were analyzed between sound and lame limbs on each day. For the MNT test, pressure tolerated by the lame limb decreased for every landmark (P < 0.05) when comparing D-1 and D+1. The sound limb tolerated more pressure on D+1 and D+6 than on baseline D-1 (P < 0.05). Thermal stimulation tolerated by the sound limb did not change over the 3 d (P > 0.05). However, the sows tolerated less heat stimulation on their lame limb on D+1 compared to D-1 levels (P < 0.05). Both MNT and TNT tests indicated greater pain sensitivity thresholds when sows were acutely lame.

Relationship between litters per sow per year sire breeding values and sire progeny means for farrowing rate, removal parity and lifetime born alive.

The objective of this study was to determine the relationship between individual sire estimated breeding values (EBV) for litters/sow/year (LSY) and sire progeny means for farrowing rate (FR), removal parity and lifetime born alive (LTBA). Genetic parameters and breeding values were estimated using ASREML. The heritability estimate for LSY was 0.11. When all sires with 10 or more daughters with records were included in the analysis, Spearman rank correlations between the sire's LSY EBV and the sires' daughter means for FR, removal parity and LTBA were 0.49, 0.23 and 0.25 (p < 0.01). The sire EBV for LSY was favourably correlated with sires' daughter means for all three traits. This provides evidence that selecting sires with high EBV for LSY could improve herd FR, removal parity and LTBA. By including LSY as part of the selection criterion, the LTBA may be indirectly improved. The positive genetic correlation between LTBA and LSY may be a result of the improved longevity of sows with greater LSY compared with sows with lower LSY. The relationships between LSY and FR, removal parity and LTBA are strongly supported by the correlations between the sire progeny means for each trait and the sire LSY EBV.

Validation of a lameness model in sows using physiological and mechanical measurements.

The objective of this study was to develop a validated, transient, chemically induced lameness model in sows using subjective and objective lameness detection tools. Experiment 1 determined an effective joint injection technique based on volume and placement of dye using feet collected from 9 finisher pigs and 10 multiparity cull sow carcasses. Experiment 2 confirmed the injection technique in live animals and produced a transient clinical lameness in 4 anesthetized sows injected with amphotericin B (15 mg/mL) in the distal interphalangeal joints of the claw. Clinical lameness was assessed by a categorical lameness scoring system, and a postmortem visual confirmation of joint injection technique was obtained. In Exp. 3, 6 sows were injected with 0, 10, or 15 mg/mL amphotericin B in either the left or right hind foot and were monitored until clinical resolution. Treated sows demonstrated elevated clinical lameness scores. These changes resolved by 7 d after lameness induction. Control sows injected with sterile saline developed a clinical lameness score of 0.5, which resolved 72 h post injection. In Exp. 4, 36 sows were injected with 10 mg/mL amphotericin B in 1 of 4 injection sites (left front claws, right front claws, left rear claws, and right rear claws). All injected sows exhibited a decrease in maximum pressure, stance time, and number of sensors activated on the GaitFour (P < 0.05) sensor system. A static force plate also demonstrated a decrease in weight (kg) being placed on the injected foot when all feet were injected (P ≤ 0.05). Injection of amphotericin B induced a predictable acute lameness that resolved spontaneously and is an effective method to model lameness in sows.

Quantitative detection of protein expression in single cells using droplet microfluidics.

We demonstrate that single cells can be controllably compartmentalized within aqueous microdroplets; using such an approach we perform high-throughput screening by detecting the expression of a fluorescent protein in individual cells with simultaneous measurement of droplet size and cell occupancy.

Pantothenate biosynthesis in higher plants.

Pantothenate (vitamin B5) is a water-soluble vitamin essential for the synthesis of CoA and ACP (acyl-carrier protein, cofactors in energy yielding reactions including carbohydrate metabolism and fatty acid synthesis. Pantothenate is synthesized de novo by plants and micro-organisms; however, animals obtain the vitamin through their diet. Utilizing our knowledge of the pathway in Escherichia coli, we have discovered and cloned genes encoding the first and last enzymes of the pathway from Arabidopsis, panB1, panB2 and panC. It is unlikely that there is a homologue of the E. coli panD gene, therefore plants must make beta-alanine by an alternative route. Possible candidates for the remaining gene, panE, are being investigated. GFP (green fluorescent protein) fusions of the three identified plant enzymes have been generated and the subcellular localization of the enzymes studied. Work is now being performed to elucidate expression patterns of the transcripts and characterize the proteins encoded by these genes.

Mechanistic and inhibition studies of chorismate-utilizing enzymes.

The shikimate biosynthetic pathway is utilized in algae, higher plants, bacteria, fungi and apicomplexan parasites; it involves seven enzymatic steps in which phosphoenolpyruvate and erythrose 4-phosphate are converted into chorismate. In Escherichia coli, five chorismate-utilizing enzymes catalyse the synthesis of aromatic compounds such as L-phenylalanine, L-tyrosine, L-tryptophan, folate, ubiquinone and siderophores such as yersiniabactin and enterobactin. As mammals do not possess such a biosynthetic system, the enzymes involved in the pathway have aroused considerable interest as potential targets for the development of antimicrobial drugs and herbicides. As an initiative to investigate the mechanism of some of these enzymes, we showed that the antimicrobial effect of (6S)-6-fluoroshikimate is the result of irreversible inhibition of 4-amino-4-deoxychorismate synthase by 2-fluorochorismate. Based on this study, a catalytic mechanism for this enzyme was proposed, in which the residue Lys-274 is involved in the formation of a covalent intermediate. In another study, Yersinia enterocolitica Irp9, which is involved in the biosynthesis of the siderophore yersiniabactin, was for the first time biochemically characterized and shown to catalyse the formation of salicylate from chorismate via isochorismate as a reaction intermediate. A three-dimensional model for this enzyme was constructed that will guide the search for potent inhibitors of salicylate formation, and hence of bacterial iron uptake.

Biophysical tools to monitor enzyme-ligand interactions of enzymes involved in vitamin biosynthesis.

Knowledge of biomolecular interactions is of importance to our understanding of biological processes such as enzyme catalysis and inhibition. Biophysical techniques enable sensitive detection and accurate characterization of binding and are therefore powerful tools in enzymology and rational drug design. The applications of NMR spectroscopy and isothermal titration calorimetry to study enzyme-ligand interactions will be discussed. Recent work on ketopantoate reductase, which catalyses an important step on the biosynthetic pathway to vitamin B5, is used to illustrate the potential of this approach.

Experiences with the shikimate-pathway enzymes as targets for rational drug design.

The background and current context of work on the shikimate-pathway enzymes as potential targets for anti-bacterial, anti-fungal and anti-parasitic drugs is reviewed. Recent work on the third enzyme of the pathway, dehydroquinase, which occurs in two structurally and mechanistically distinct forms, is used to illustrate the present state of studies into rational drug design.

Structural insights into the evolution of the pantothenate-biosynthesis pathway.

Pantothenate is synthesized in bacteria, fungi and plants, and as vitamin B5 is a dietary requirement in animals. The three-dimensional structures of the four Escherichia coli enzymes involved in the production of pantothenate have been determined. We describe the use of comparative analyses of the sequences and structures to identify distant homologues of the four enzymes in an attempt to understand the evolution of the pathway. We conclude that it is likely to have evolved via a patchwork mechanism, whereby the individual enzymes were recruited separately.

Substrate deactivation of phenylalanine-sensitive 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase by erythrose 4-phosphate.

3-Deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAH7PS, EC 4.1.2.15) catalyzes the condensation of phosphoenolpyruvate (PEP) with erythrose 4-phosphate (E4P) to give DAH7P via an ordered sequential mechanism. In the absence of PEP (the first substrate to bind), E4P binds covalently to the phenylalanine-sensitive DAH7PS of Escherichia coli, DAH7PS(Phe), deactivating the enzyme. Activity is restored on addition of excess PEP but not if deactivation was carried out in the presence of sodium cyanoborohydride. Electrospray mass spectrometry indicates that a single E4P is bound to the protein. These data are consistent with a slow, reversible Schiff base reaction of the aldehydic functionality of E4P with a buried lysine. Molecular modeling indicates that Lys186, a residue at the base of the substrate-binding cavity involved in hydrogen bonding with PEP, is well placed to react with E4P forming an imine linkage that is substantially protected from solvent water.

Crystal structure of Escherichia coli ketopantoate reductase at 1.7 A resolution and insight into the enzyme mechanism.

Ketopantoate reductase (KPR, EC 1.1.1.169) catalyzes the NADPH-dependent reduction of ketopantoate to pantoate on the pantothenate (vitamin B(5)) biosynthetic pathway. The Escherichia coli panE gene encoding KPR was cloned and expressed at high levels as the native and selenomethionine-substituted (SeMet) proteins. Both native and SeMet recombinant proteins were purified by three chromatographic steps, to yield pure proteins. The wild-type enzyme was found to have a K(M)(NADPH) of 20 microM, a K(M)(ketopantoate) of 60 microM, and a k(cat) of 40 s(-1). Regular prismatic KPR crystals were prepared using the hanging drop technique. They belonged to the tetragonal space group P4(2)2(1)2, with cell parameters: a = b = 103.7 A and c = 55.7 A, accommodating one enzyme molecule per asymmetric unit. The structure of KPR was determined by the multiwavelength anomalous dispersion method using the SeMet protein, for which data were collected to 2.3 A resolution. The native data were collected to 1.7 A resolution and used to refine the final structure. The secondary structure comprises 12 alpha-helices, three 3(10)-helices, and 11 beta-strands. The enzyme is monomeric and has two domains separated by a cleft. The N-terminal domain has an alphabeta-fold of the Rossmann type. The C-terminal domain (residues 170-291) is composed of eight alpha-helices. KPR is shown to be a member of the 6-phosphogluconate dehydrogenase C-terminal domain-like superfamily. A model for the ternary enzyme-NADPH-ketopantoate ternary complex provides a rationale for kinetic data reported for specific site-directed mutants.

Direct and quantitative detection of bacteriophage by "hearing" surface detachment using a quartz crystal microbalance.

We show that it is possible to detect specifically adsorbed bacteriophage directly by breaking the interactions between proteins displayed on the phage coat and ligands immobilized on the surface of a quartz crystal microbalance (QCM). This is achieved through increasing the amplitude of oscillation of the QCM surface and sensitively detecting the acoustic emission produced when the bacteriophage detaches from the surface. There is no interference from nonspecifically adsorbed phage. The detection is quantitative over at least 5 orders of magnitude and is sensitive enough to detect as few as 20 phage. The method has potential as a sensitive and low-cost method for virus detection.

Direct and sensitive detection of a human virus by rupture event scanning.

We have developed a sensitive, economical method that directly detects viruses by making use of the interaction between type 1 herpes simplex virus (HSV1) and specific antibodies covalently attached to the oscillating surface of a quartz crystal microbalance (QCM). The virions were detached from the surface by monotonously increasing the amplitude of oscillation of the QCM, while using the QCM to sensitively detect the acoustic noise produced when the interactions were broken. We term this process rupture event scanning (REVS). The method is quantitative over at least six orders of magnitude, and its sensitivity approaches detection of a single virus particle.

Nonlinear analysis of partial dopamine agonist effects on cAMP in C6 glioma cells.

Most drugs have some efficacy so that improved methods to determine the relative intrinsic efficacy of partial agonists should be of benefit to preclinical and clinical investigators. We examined the effects of partial D(1) or partial D(2) dopamine agonists using a partial agonist interaction model. The dependent variable was the modulation of the dopamine-receptor-mediated cAMP response in C6 glioma cells selectively and stably expressing either D(1) or D(2) recombinant dopamine receptors. The dissociation constant (K(B)) and relative intrinsic efficacy (E(r)) for each partial agonist were calculated using a partial agonist interaction null model in which the effects of fixed concentrations of each partial agonist on the dopamine dose-response curve were evaluated. This model is an extension of the competitive antagonist null model to drugs with efficacy and assumes only that the log-dose--response curve is monotonic. Generally, the partial agonist interaction model fit the data, as well as fits of the independent logistic curves. Furthermore, the partial agonist K(B) values could be shared across partial agonist concentrations without worsening the model fit (by increasing the residual variance). K(B) values were also similar to drug affinities reported in the literature. The model was validated in three ways. First, we assumed a common tissue stimulus parameter (beta) and calculated the E(r) values. This provided a qualitative check on the interaction model results. Second, we calculated new relative efficacy values, E(r)(beta), using the beta estimate. Third, we calculated relative efficacy using relative maxima times midpoint shift ratios (J. Theor. Biol. 198 (1999) 347.). All three methods indicated that the present model yielded reasonable estimates of affinity and relative efficacy for the set of compounds studied. Our results provide a quick and convenient method of quantification of partial agonist efficacy. Special applications and limitations of the model are discussed. In addition, the present results are the first report of the relative intrinsic efficacy values for this set of D(2) ligands.

Novel chemical tagging method for combinatorial synthesis utilizing suzuki chemistry and fourier transform ion cyclotron resonance mass spectrometry.

We describe a novel chemical tagging strategy for combinatorial solid-phase chemistry. The tags used are para-substituted alkyl phenols, with the first tags attached directly to the chloromethyl polystyrene and subsequent tags attached via Suzuki couplings using either aryl diboronic acids or aryl iodides. The identities of the tags attached to a single bead are discovered by the high-resolution, accurate mass technique of Fourier transform ion cyclotron resonance mass spectrometry. The method is exemplified for the coded assembly of a tripeptide.

The crystal structure of E. coli pantothenate synthetase confirms it as a member of the cytidylyltransferase superfamily.

BACKGROUND: Pantothenate synthetase (EC 6.3.2.1) is the last enzyme of the pathway of pantothenate (vitamin B(5)) synthesis. It catalyzes the condensation of pantoate with beta-alanine in an ATP-dependent reaction. RESULTS: We describe the overexpression, purification, and crystal structure of recombinant pantothenate synthetase from E. coli. The structure was solved by a selenomethionine multiwavelength anomalous dispersion experiment and refined against native data to a final R(cryst) of 22.6% (R(free) = 24.9%) at 1.7 A resolution. The enzyme is dimeric, with two well-defined domains per protomer: the N-terminal domain, a Rossmann fold, contains the active site cavity, with the C-terminal domain forming a hinged lid. CONCLUSIONS: The N-terminal domain is structurally very similar to class I aminoacyl-tRNA synthetases and is thus a member of the cytidylyltransferase superfamily. This relationship has been used to suggest the location of the ATP and pantoate binding sites and the nature of hinge bending that leads to the ternary enzyme-pantoate-ATP complex.