Large Animal Translational Validation of 3 Mitral Valve Repair Operations for Mitral Regurgitation Using a Mitral Valve Prolapse Model: A Comprehensive In Vivo Biomechanical Engineering Analysis.

BACKGROUND: Various mitral repair techniques have been described. Though these repair techniques can be highly effective when performed correctly in suitable patients, limited quantitative biomechanical data are available. Validation and thorough biomechanical evaluation of these repair techniques from translational large animal in vivo studies in a standardized, translatable fashion are lacking. We sought to evaluate and validate biomechanical differences among different mitral repair techniques and further optimize repair operations using a large animal mitral valve prolapse model. METHODS: Male Dorset sheep (n=20) had P2 chordae severed to create the mitral valve prolapse model. Fiber Bragg grating force sensors were implanted to measure chordal forces. Ten sheep underwent 3 randomized, paired mitral valve repair operations: neochord repair, nonresectional leaflet remodeling, and triangular resection. The other 10 sheep underwent neochord repair with 2, 4, and 6 neochordae. Data were collected at baseline, mitral valve prolapse, and after each repair. RESULTS: All mitral repair techniques successfully eliminated regurgitation. Compared with mitral valve prolapse (0.54±0.18 N), repair using neochord (0.37±0.20 N; P=0.02) and remodeling techniques (0.30±0.15 N; P=0.001) reduced secondary chordae peak force. Neochord repair further decreased primary chordae peak force (0.21±0.14 N) to baseline levels (0.20±0.17 N; P=0.83), and was associated with lower primary chordae peak force compared with the remodeling (0.34±0.18 N; P=0.02) and triangular resectional techniques (0.36±0.27 N; P=0.03). Specifically, repair using 2 neochordae resulted in higher peak primary chordal forces (0.28±0.21 N) compared with those using 4 (0.22±0.16 N; P=0.02) or 6 neochordae (0.19±0.16 N; P=0.002). No difference in peak primary chordal forces was observed between 4 and 6 neochordae (P=0.05). Peak forces on the neochordae were the lowest using 6 neochordae (0.09±0.11 N) compared with those of 4 neochordae (0.15±0.14 N; P=0.01) and 2 neochordae (0.29±0.18 N; P=0.001). CONCLUSIONS: Significant biomechanical differences were observed underlying different mitral repair techniques in a translational large animal model. Neochord repair was associated with the lowest primary chordae peak force compared to the remodeling and triangular resectional techniques. Additionally, neochord repair using at least 4 neochordae was associated with lower chordal forces on the primary chordae and the neochordae. This study provided key insights about mitral valve repair optimization and may further improve repair durability.

Strongly Polarized π-Extended 1,4-Dihydropyrrolo[3,2-b]pyrroles Fused with Tetrazolo[1,5-a]quinolines.

A straightforward route to 1,4-dihydropyrrolo[3,2-b]pyrroles comprised of two electron-withdrawing quinoline or tetrazolo[1,5-a]quinoline scaffolds has been developed. The versatile multicomponent reaction affording 1,4-dihydropyrrolo[3,2-b]pyrroles combined with intramolecular direct arylation enables assembly of these products in just three steps from anilines with overall yields exceeding 30%. The planarized, ladder-type heteroacenes possess up to 14 conjugated rings. These nominally quadrupolar materials exhibit efficient fluorescence with wavelengths spanning most of the visible spectrum from green-yellow for the dyes possessing biaryl bridges and orange-red for the fully fused systems. In many cases, the fluorescence quantum yields are large, the solvatofluorochromic effects are strong, and the fluorescence is maintained even in crystalline state. Analysis of the electronic structure of these molecular architectures using quantum chemical methods suggests that the character and position of the flanking heterocycle determine the shape of HOMO and LUMO and their extension to N-aryl substituents, influencing the values of molar absorption coefficient. An experimental study of the two-photon absorption (2PA) properties has revealed that it occurs in the 700-800 nm range with apparent deviation from the Laporte parity selection rule, which may be attributed to Hertzberg-Teller contribution to vibronically allowed 2PA transition.

Evaluation of Truck Cab Decontamination Procedures following Inoculation with Porcine Epidemic Diarrhea Virus and Porcine Reproductive and Respiratory Syndrome Virus.

This experiment aimed to evaluate commercially available disinfectants and their application methods against porcine epidemic diarrhea virus (PEDV) and porcine reproductive and respiratory syndrome virus (PRRSV) on truck cab surfaces. Plastic, fabric, and rubber surfaces inoculated with PEDV or PRRSV were placed in a full-scale truck cab and then treated with one of eight randomly assigned disinfectant treatments. After application, surfaces were environmentally sampled with cotton gauze and tested for PEDV and PRRSV using qPCR duplex analysis. There was a disinfectant × surface interaction (p < 0.0001), indicating a detectable amount of PEDV or PRRSV RNA was impacted by disinfectant treatment and surface material. For rubber surfaces, 10% bleach application had lower detectable amounts of RNA compared to all other treatments (p < 0.05) except Intervention via misting fumigation, which was intermediate. In both fabric and plastic surfaces, there was no evidence (p > 0.05) of a difference in detectable RNA between disinfectant treatments. For disinfectant treatments, fabric surfaces with no chemical treatment had less detectable viral RNA compared to the corresponding plastic and rubber (p < 0.05). Intervention applied via pump sprayer to fabric surfaces had less detectable viral RNA than plastic (p < 0.05). Furthermore, 10% bleach applied via pump sprayer to fabric and rubber surfaces had less detectable viral RNA than plastic (p < 0.05). Also, a 10 h downtime, with no chemical application or gaseous fumigation for 10 h, applied to fabric surfaces had less detectable viral RNA than other surfaces (p < 0.05). Sixteen treatments were evaluated via swine bioassay, but all samples failed to produce infectivity. In summary, commercially available disinfectants successfully reduced detectable viral RNA on surfaces but did not eliminate viral genetic material, highlighting the importance of bioexclusion of pathogens of interest.

Biomechanical engineering analysis of neochordae length's impact on chordal forces in mitral repair.

OBJECTIVES: Artificial neochordae implantation is commonly used for mitral valve (MV) repair. However, neochordae length estimation can be difficult to perform. The objective was to assess the impact of neochordae length changes on MV haemodynamics and neochordal forces. METHODS: Porcine MVs (n = 6) were implanted in an ex vivo left heart simulator. MV prolapse (MVP) was generated by excising at least 2 native primary chordae supporting the P2 segments from each papillary muscle. Two neochordae anchored on each papillary muscle were placed with 1 tied to the native chord length (exact length) and the other tied with variable lengths from 2× to 0.5× of the native length (variable length). Haemodynamics, neochordal forces and echocardiography data were collected. RESULTS: Neochord implantation repair successfully eliminated mitral regurgitation with repaired regurgitant fractions of approximately 4% regardless of neochord length (P < 0.01). Leaflet coaptation height also significantly improved to a minimum height of 1.3 cm compared with that of MVP (0.9 ± 0.4 cm, P < 0.05). Peak and average forces on exact length neochordae increased as variable length neochordae lengths increased. Peak and average forces on the variable length neochordae increased with shortened lengths. Overall, chordal forces appeared to vary more drastically in variable length neochordae compared with exact length neochordae. CONCLUSIONS: MV regurgitation was eliminated with neochordal repair, regardless of the neochord length. However, chordal forces varied significantly with different neochord lengths, with a preferentially greater impact on the variable length neochord. Further validation studies may be performed before translating to clinical practices.

Effect of the pelleting process on diet formulations with varying levels of crystalline amino acids and reducing sugars on digestibility in growing pigs.

The objective of this experiment was to determine the effects of pelleting on the standardized ileal digestibility (SID) of amino acids (AA) and crude protein (CP) in diets with or without increased concentrations of free AA and reducing sugars (RS). Eight individually housed, ileal cannulated barrows (initially 31.4 kg) were allotted to an 8 × 8 Latin square with eight diets and eight 7-d periods with ileal digesta collected on days 6 and 7. Treatments were arranged in a 2 × 2 × 2 factorial with the main effects of diet form (mash or pellet), crystalline AA (low or high), or RS (low or high), provided by distillers dried grains with solubles and bakery meal. Diets were pelleted to achieve a hot pellet temperature of 85 to 88 °C. Data were analyzed as a Latin square design using the GLIMMIX procedure of SAS 9.4. A feed form × RS interaction (P < 0.026) for SID of tryptophan was observed. Feeding pelleted low RS diets increased SID of tryptophan compared with mash high and low RS diets, and pelleted high RS diets. For the main effects of feed form, the SID of total AA, CP, and indispensable AA was greater (P < 0.042) in pelleted diets compared with mash diets. For the main effects of crystalline AA, pigs fed high crystalline AA had increased (P = 0.007) SID of tryptophan and decreased (P = 0.050) SID of histidine compared with those fed low crystalline AA diets. For the main effects of RS, high RS diets had decreased (P < 0.05) SID of total AA, CP, and indispensable AA compared with low RS diets. In conclusion, pelleting diets increased AA digestibility, and pelleting diets with increased crystalline AA or RS did not affect the improvement in AA digestibility from pelleting. Diets formulated with high crystalline AA had increased SID of tryptophan. Formulating diets with high RS resulted in decreased AA digestibility compared with corn-soybean meal-based diets.

The objective of this study was to determine the effects of pelleting on the standardized ileal digestibility (SID) of amino acids (AA) in diets with or without increased concentrations of free AA and reducing sugars (RS). Treatments were arranged in a 2 × 2 × 2 factorial with the main effects of diet form (mash or pellet), crystalline AA (low or high), or RS (low or high), provided by dried distillers grains with solubles and bakery meal. A total of 8 illeal cannulated barrows were fed treatments in an 8 × 8 Latin square design. Results indicated that there was no evidence of interactions between diet types and diet form, indicating that increasing amounts of crystalline AA and RS did not reduce amino acid digestibility when pelleting diets. Additionally, pelleting diets resulted in increased amino acid digestibility compared to mash diets. Diets formulated with 20% dried distillers grains with solubles and 15% bakery resulted in decreased amino acid digestibility compared with the corn­soybean meal-based diets. Crystalline amino acid concentration did not influence amino acid digestibility of indispensable AA, except for SID of tryptophan which was increased in diets with higher concentrations of crystalline AA.

Chordal force profile after neochordal repair of anterior mitral valve prolapse: An ex vivo study.

Objective: This study aimed to biomechanically evaluate the force profiles on the anterior primary and secondary chordae after neochord repair for anterior valve prolapse with varied degrees of residual mitral regurgitation using an ex vivo heart simulator. Methods: The experiment used 8 healthy porcine mitral valves. Chordal forces were measured using fiber Bragg grating sensors on primary and secondary chordae from A2 segments. The anterior valve prolapse model was generated by excising 2 primary chordae at the A2 segment. Neochord repair was performed with 2 pairs of neochords. Varying neochord lengths simulated postrepair residual mitral regurgitation with regurgitant fraction at >30% (moderate), 10% to 30% (mild), and <10% (perfect repair). Results: Regurgitant fractions of baseline, moderate, mild, and perfect repair were 4.7% ± 0.8%, 35.8% ± 2.1%, 19.8% ± 2.0%, and 6.0% ± 0.7%, respectively (P < .001). Moderate had a greater peak force of the anterior primary chordae (0.43 ± 0.06 N) than those of baseline (0.19 ± 0.04 N; P = .011), mild (0.23 ± 0.05 N; P = .041), and perfect repair (0.21 ± 0.03 N; P = .006). In addition, moderate had a greater peak force of the anterior secondary chordae (1.67 ± 0.17 N) than those of baseline (0.64 ± 0.13 N; P = .003), mild (0.84 ± 0.24 N; P = .019), and perfect repair (0.68 ± 0.14 N; P = .001). No significant differences in peak and average forces on both primary and secondary anterior chordae were observed between the baseline and perfect repair as well as the mild and perfect repair. Conclusions: Moderate residual mitral regurgitation after neochord repair was associated with increased anterior primary and secondary chordae forces in our ex vivo anterior valve prolapse model. This difference in chordal force profile may influence long-term repair durability, providing biomechanical evidence in support of obtaining minimal regurgitation when repairing mitral anterior valve prolapse.

Biomechanics and clinical outcomes of various conduit configurations in valve sparing aortic root replacement.

Background: Several conduit configurations, such as straight graft (SG), Valsalva graft (VG), anticommissural plication (ACP), and the Stanford modification (SMOD) technique, have been described for the valve-sparing aortic root replacement (VSARR) procedure. Prior ex vivo studies have evaluated the impact of conduit configurations on root biomechanics, but the mock coronary artery circuits used could not replicate the physical properties of native coronary arteries. Moreover, the individual leaflet's biomechanics, including the fluttering phenomenon, were unclear. Methods: Porcine aortic roots with coronary arteries were explanted (n=5) and underwent VSARR using SG, VG, ACP, and SMOD for evaluation in an ex vivo left heart flow loop simulator. Additionally, 762 patients who underwent VSARR from 1993 through 2022 at our center were retrospectively reviewed. Analysis of variance was performed to evaluate differences between different conduit configurations, with post hoc Tukey's correction for pairwise testing. Results: SG demonstrated lower rapid leaflet opening velocity compared with VG (P=0.001) and SMOD (P=0.045) in the left coronary cusp (LCC), lower rapid leaflet closing velocity compared with VG (P=0.04) in the right coronary cusp (RCC), and lower relative opening force compared with ACP (P=0.04) in the RCC. The flutter frequency was lower in baseline compared with VG (P=0.02) and in VG compared with ACP (P=0.03) in the LCC. Left coronary artery mean flow was higher in SG compared with SMOD (P=0.02) and ACP (P=0.05). Clinically, operations using SG compared with sinus-containing graft was associated with shorter aortic cross-clamp and cardiopulmonary bypass time (P<0.001, <0.001). Conclusions: SG demonstrated hemodynamics and biomechanics most closely recapitulating those from the native root with significantly shorter intraoperative times compared with repair using sinus-containing graft. Future in vivo validation studies as well as correlation with comprehensive, comparative clinical study outcomes may provide additional invaluable insights regarding strategies to further enhance repair durability.

Optimization of Freeform Reversible Embedding of Suspended Hydrogel Microspheres for Substantially Improved Three-Dimensional Bioprinting Capabilities.

Three-dimensional (3D) bioprinting demonstrates technology that is capable of producing structures comparable to native tissues in the human body. The freeform reversible embedding of suspended hydrogels (FRESH) technique involves hydrogel-based bio-inks printed within a thermo-reversible support bath to provide mechanical strength to the printed construct. Smaller and more uniform microsphere sizes of FRESH were reported to aid in enhancing printing resolution and construct accuracy. Therefore, we sought to optimize the FRESH generation protocol, particularly by varying stir speed and stir duration, in hopes to further improve microsphere size and uniformity. We observed optimal conditions at a stir speed of 600 rpm and stir duration for 20 h that generated the smallest microspheres with the best uniformity. Comparison of using the optimized FRESH to the commercial FRESH LifeSupport to bioprint single filament and geometrical constructs revealed reduced single filament diameters and higher angular precision in the optimized FRESH bio-printed constructs compared with those printed in the commercial FRESH. Overall, our refinement of the FRESH manufacturing protocol represents an important step toward enhancing 3D bioprinting resolution and construct fidelity. Improving such technologies allows for the fabrication of highly accurate constructs with anatomical properties similar to native counterparts. Such work has significant implications in the field of tissue engineering for producing accurate human organ model systems. Impact statement Freeform reversible embedding of suspended hydrogels (FRESH) is a method of sacrificial three-dimensional (3D) bioprinting that offers support to reinforce bio-ink extrusion during printing. During FRESH generation, the stir speed and stir duration of the mixture can significantly impact FRESH microsphere characteristics. In this study, we optimized FRESH microspheres to significantly improve resolution and accuracy in bioprinting. This advancement in FRESH-based 3D bioprinting technologies allows for the fabrication of highly accurate constructs with anatomical properties similar to native counterparts and has significant implications in the field of tissue engineering and translational medicine.

Three-Dimensional Bioprinting with Alginate by Freeform Reversible Embedding of Suspended Hydrogels with Tunable Physical Properties and Cell Proliferation.

Extrusion-based three-dimensional (3D) bioprinting is an emerging technology that allows for rapid bio-fabrication of scaffolds with live cells. Alginate is a soft biomaterial that has been studied extensively as a bio-ink to support cell growth in 3D constructs. However, native alginate is a bio-inert material that requires modifications to allow for cell adhesion and cell growth. Cells grown in modified alginates with the RGD (arginine-glycine-aspartate) motif, a naturally existing tripeptide sequence that is crucial to cell adhesion and proliferation, demonstrate enhanced cell adhesion, spreading, and differentiation. Recently, the bioprinting technique using freeform reversible embedding of suspended hydrogels (FRESH) has revolutionized 3D bioprinting, enabling the use of soft bio-inks that would otherwise collapse in air. However, the printability of RGD-modified alginates using the FRESH technique has not been evaluated. The associated physical properties and bioactivity of 3D bio-printed alginates after RGD modification remains unclear. In this study, we characterized the physical properties, printability, and cellular proliferation of native and RGD-modified alginate after extrusion-based 3D bioprinting in FRESH. We demonstrated tunable physical properties of native and RGD-modified alginates after FRESH 3D bioprinting. Sodium alginate with RGD modification, especially at a high concentration, was associated with greatly improved cell viability and integrin clustering, which further enhanced cell proliferation.

Impact of storage conditions and premix type on water-soluble vitamin stability.

Mitigation options to reduce the risk of foreign animal disease entry into the United States may lead to degradation of some vitamins. The objective of Exp. 1 was to determine the impact of 0, 30, 60, or 90 d storage time on water-soluble vitamin (riboflavin, niacin, pantothenic acid, and cobalamin) stability when vitamin premix (VP) and vitamin trace mineral premix (VTM) were blended with 1% inclusion of medium-chain fatty acid (MCFA) (1:1:1 blend of C6:C8:C10) or mineral oil (MO) with different environmental conditions. Samples stored at room temperature (RT) (approximately 22 °C) or in an environmentally controlled chamber set at 40 °C and 75% humidity, high temperature high humidity (HTHH). The sample bags were pulled out at day 0, 30, 60, and 90 for RT condition and HTHH condition. Therefore, treatments were analyzed as a 2 × 2 × 2 × 3 factorial, with two premix types (vitamin premix vs. VTM), two oil types (MO vs. MCFA), two storage conditions (RT vs. HTHH), and three time points (day 30, 60, and 90). The objective of Exp. 2 was to determine the effect of heat pulse treatment and MCFA addition on water-soluble vitamin (riboflavin, niacin, pantothenic acid, and cobalamin) stability with two premix types. A sample from each treatment was heated at 60 °C and 20% humidity. Therefore, treatments were analyzed as a 2 × 2 factorial, with two premix types (VP vs. VTM) and two oil types (MO vs. MCFA). For Exp. 1, the following effects were significant for riboflavin: main effect of premix type (P < 0.0001), storage condition (P = 0.015), and storage time (P < 0.0001); for pantothenic acid: premix type × storage time × storage condition (P = 0.003) and premix type × oil type (P < 0.0001) interactions; and for cobalamin: premix type × storage condition (P < 0.0001) and storage time × storage condition (P < 0.0001) interactions and main effect of oil type (P = 0.018). The results of Exp. 2 demonstrated that there was an interaction between oil type and premix type for only pantothenic acid (P = 0.021). The oil type did not affect the stability of riboflavin, niacin, or cobalamin and pantothenic acid stability was not different within similar premixes. The only difference in water-soluble vitamin stability between VP and VTM was for pantothenic acid (P < 0.001). The results of this experiment demonstrated that the stability of water soluble vitamins are dependent on the vitamin of interest and the conditions at which it is stored.

On-off-on Control of Molecular Inversion Symmetry via Multi-stage Protonation: Elucidating Vibronic Laporte Rule.

The Laporte rule dictates that one- and two-photon absorption spectra of inversion-symmetric molecules should display alternatively forbidden electronic transitions; however, for organic fluorophores, drawing clear distinction between the symmetric- and non-inversion symmetric two-photon spectra is often obscured due to prevalent vibronic interactions. We take advantage of consecutive single- and double-protonation to break and then reconstitute inversion symmetry in a nominally symmetric diketopyrrolopyrrole, causing large changes in two-photon absorption. By performing detailed one- and two-photon titration experiments, with supporting quantum-chemical model calculations, we explain how certain low-frequency vibrational modes may lead to apparent deviations from the strict Laporte rule. As a result, the system may be indeed considered as an on-off-on inversion symmetry switch, opening new avenues for two-photon sensing applications.

A novel photosynthetic biologic topical gel for enhanced localized hyperoxygenation augments wound healing in peripheral artery disease.

Peripheral artery disease and the associated ischemic wounds are substantial causes of global morbidity and mortality, affecting over 200 million people worldwide. Although advancements have been made in preventive, pharmacologic, and surgical strategies to treat this disease, ischemic wounds, a consequence of end-stage peripheral artery disease, remain a significant clinical and economic challenge. Synechococcus elongatus is a cyanobacterium that grows photoautotrophically and converts carbon dioxide and water into oxygen. We present a novel topical biologic gel containing S. elongatus that provides oxygen via photosynthesis to augment wound healing by rescuing ischemic tissues caused by peripheral artery disease. By using light rather than blood as a source of energy, our novel topical therapy significantly accelerated wound healing in two rodent ischemic wound models. This novel topical gel can be directly translated to clinical practice by using a localized, portable light source without interfering with patients' daily activities, demonstrating potential to generate a paradigm shift in treating ischemic wounds from peripheral artery disease. Its novelty, low production cost, and ease of clinical translatability can potentially impact the clinical care for millions of patients suffering from peripheral arterial disease.

Using caloric efficiency to estimate the net energy value of expelled, extruded soybean meal relative to dehulled, solvent-extracted soybean meal and its effects on growth performance of nursery pigs.

This study aimed to estimate the net energy (NE) value of expelled, extruded soybean meal (MSBM) relative to dehulled, solvent-extracted soybean meal (SSBM) and determine its effects on growth performance of late nursery pigs. A total of 297 pigs (DNA 241 × 600) were weaned (BW 5.10 kg) and placed into 60 pens (2 rooms of 30 pens) with 5 pigs per pen balanced by gender and weaning weight. Pigs were fed a common diet for 21 d. Then, pens of pigs (BW 9.3 kg) were randomly assigned to one of five treatments to provide 12 replications per treatment. Treatments consisted of increasing amounts of MSBM replacing SSBM in the diet (0%, 25%, 50%, 75%, and 100%). All diets were fed for 28 d and were formulated to 1.30% standardized ileal digestible lysine and met or exceeded requirements for amino acids, calcium, and phosphorus. The SSBM diet was formulated to 2,421 kcal/kg and NE was not balanced between diets. Analyzed values for CP, EE, CF, and total lysine for the SSBM were 47.28%, 0.47%, 3.80%, and 3.00%, whereas the MSBM contained 47.41%, 6.88%, 5.32%, and 2.99%, respectively. The MSBM had increased values for KOH solubility and trypsin inhibitor (83.62% and 7,026 TIU/g) compared to the SSBM (73.05% and 3,011 TIU/g), whereas urease activity was similar between the two (0.03 and 0.02 Δ pH, respectively). Data were analyzed using Proc GLIMMIX (SAS 9.4; Cary, NC) with pen as the experimental unit and room as the blocking factor. There was no evidence of differences in ADG and ADFI in pigs fed diets with increasing concentrations of MSBM. Pigs fed diets with increasing concentrations of MSBM had improved (linear, P < 0.001) G:F and caloric efficiency on an NE basis. Using caloric efficiency to estimate NE of the MSBM relative to SSBM, MSBM was estimated to have a value of 2,566 kcal/kg. In conclusion, MSBM contains approximately 123% of the energy of SSBM, which improved feed efficiency when fed to nursery pigs.

Effect of mixing and feed batch sequencing on the prevalence and distribution of African swine fever virus in swine feed.

It is critical to have methods that can detect and mitigate the risk of African swine fever virus (ASFV) in potentially contaminated feed or ingredients bound for the United States. The purpose of this work was to evaluate feed batch sequencing as a mitigation technique for ASFV contamination in a feed mill, and to determine if a feed sampling method could identify ASFV following experimental inoculation. Batches of feed were manufactured in a BSL-3Ag room at Kansas State University's Biosafety Research Institute in Manhattan, Kansas. First, the pilot feed manufacturing system mixed, conveyed, and discharged an ASFV-free diet. Next, a diet was manufactured using the same equipment, but contained feed inoculated with ASFV for final concentration of 5.6 × 104 TCID50 /g. Then, four subsequent ASFV-free batches of feed were manufactured. After discharging each batch into a collection container, 10 samples were collected in a double 'X' pattern. Samples were analysed using a qPCR assay for ASFV p72 gene then the cycle threshold (Ct) and Log10 genomic copy number (CN)/g of feed were determined. The qPCR Ct values (p < .0001) and the Log10 genomic CN/g (p < .0001) content of feed samples were impacted based on the batch of feed. Feed samples obtained after manufacturing the ASFV-contaminated diet contained the greatest amounts of ASFV p72 DNA across all criteria (p < .05). Quantity of ASFV p72 DNA decreased sequentially as additional batches of feed were manufactured, but was still detectable after batch sequence 4. This subsampling method was able to identify ASFV genetic material in feed samples using p72 qPCR. In summary, sequencing batches of feed decreases concentration of ASFV contamination in feed, but does not eliminate it. Bulk ingredients can be accurately evaluated for ASFV contamination by collecting 10 subsamples using the sampling method described herein. Future research is needed to evaluate if different mitigation techniques can reduce ASFV feed contamination.

Novel Lipophilic Fluorophores with Highly Acidity-Dependent Two-Photon Response.

Lipophilic fluorophores are widely implemented in nonlinear microscopy; however, few existing membrane-specific probes combine the high brightness of two-photon excited fluorescence (2PEF) with pH sensitivity. Herein we describe four novel two-photon excited fluorophores, based on a coumarin 151 core structure, where lipophilicity is induced by a covalently attached phosphazene moiety. Changing the environmental acidity using trifluoromethanesulfonic (triflic) acid leads to profound changes in the linear fluorescence and 2PEF characteristics, due to chromophores' switching between neutral- and protonated forms. We characterize this dependence by measuring the two-photon absorption (2PA) spectra over the region λ2PA =550-1000 nm, observing 2PA cross sections of σ2PA =10-20 GM, with an associated 2PEF brightness of 10-13 GM, in neutral solutions of both acetonitrile and n-octanol. Although quantum chemical modelling and NMR measurements show that, at high chromophore concentrations, protonation may be accompanied by a dimerization process, these dimers likely do not form at the lower concentrations used in optical spectroscopy.

Effects of conditioning temperature and pellet mill die speed on pellet quality and relative stabilities of phytase and xylanase.

The objective of this experiment was to determine the effect of conditioning temperature and die speed on pellet quality and enzyme stability of phytase and xylanase. Treatments were initially arranged as a 2 × 3 factorial of conditioning temperature (74 and 85 °C) and die speed (127, 190, and 254 rpm); however, when conditioning at 85 °C, it was not possible to pellet at 127 rpm. Thus, data were analyzed in two different segments using the GLIMMIX procedure of SAS. First, linear and quadratic contrasts were utilized to test the response to increasing die speed at 74 °C. Second, the data was analyzed as a 2 × 2 factorial of conditioning temperature (74 and 85 °C) and die speed (190 and 254 rpm). Treatments were arranged in a completely randomized design and replicated three times. Diets were conditioned for approximately 30 s and pelleted with a 4.8-mm-diameter × 44.5-mm-effective length die at a rate of 4.5 MT/h. Pellet durability index (PDI) was determined using the tumble box and Holmen NHP 100 methods. Samples of the unconditioned mash (M), conditioned mash (CM), and pellets (P) were collected and analyzed for phytase and xylanase concentration. Relative enzyme stabilities were expressed as CM:M, P:CM, and P:M. Stabilities expressed as P:M were used an indication of enzyme stability through the entire pelleting process. Diets conditioned at 74 °C showed no evidence of difference in phytase or xylanase P:M stability when decreasing die speed from 254 to 127 rpm. However, when conditioning diets at 74 °C, decreasing die speed increased (linear, P < 0.001) PDI. There was no conditioning temperature × die speed interaction for overall xylanase P:M stability or PDI. However, there was a conditioning temperature × die speed interaction (P < 0.01) for phytase P:M stability. When conditioning diets at 85 °C, increasing die speed decreased phytase P:M stability. However, when conditioning at 74 °C, increasing die speed did not influence phytase P:M stability. For main effects of conditioning temperature, increasing temperature improved (P < 0.001) PDI with no evidence of difference for xylanase P:M stability. For the main effects of die speed (254 vs. 190 rpm), decreasing die speed decreased (P < 0.001) the P:M xylanase stability, but there was no evidence of difference for PDI. The results of this trial indicate that die speed should be taken into consideration when evaluating enzyme stability of both phytase and xylanase as pellet mill models may be operating at different speeds. Additionally, increasing conditioning temperature will improve PDI but may result in decreased phytase stability.

Effect of cleaning corn on mycotoxin concentration and nursery pig growth performance.

Mycotoxins are naturally produced hazards that result from molds grown on cereal grains and other commodities. These molds may produce carcinogenic mycotoxins, which can be harmful to humans and animals. Removing broken kernels has been demonstrated to reduce mycotoxin concentration, but with high variability. Therefore, two experiments were conducted to quantify the magnitude of natural mycotoxin concentration that may be reduced by cleaning corn. Two loads of corn that were naturally contaminated with mycotoxins were procured. Corn for Experiment 1 was contaminated with aflatoxin (1,074 parts per billion; ppb), fumonisin (8.3 parts per million; ppm), and ochratoxin A (206 ppb), while corn for Experiment 2 was contaminated with only fumonisin (5.5 ppm). Corn was cleaned by mechanical sieving. For each experiment, corn was divided into twenty 150 kg runs. Runs were randomly assigned to 1 of 4 experimental treatments: 1) no screen 2) 12.7 mm screen, 3) 4.8 mm screen, and 4) 12.7 + 4.8-mm screen. The corn cleaner was sanitized between runs. Three 5 kg corn samples were collected from each run, and analyzed for mycotoxin concentration. In Experiment 1, cleaning reduced (P < 0.05) aflatoxin and fumonisin concentration by an average of 26% and 45%, respectively, compared to the original uncleaned corn level, but did not impact (P > 0.10) ochratoxin A. The resultant screenings had nearly four times the aflatoxin (4,224 ppb) and 7.5 times the fumonisin concentration (60.4 ppm) as the uncleaned corn. In Experiment 2, cleaning reduced (P < 0.05) fumonisin concentration by 32%. The resultant screenings had 19.6 times the fumonisin concentration (65.4 ppm) as the uncleaned corn. To determine the effect that cleaning corn may have on nursery pig growth performance, 360 nursery pigs were used in Experiment 3 to evaluate the impact of cleaning or pelleting on growth performance. Treatments were arranged in a 2 × 3 factorial with corn type (uncleaned vs. cleaned) and feed form (mash vs. pelleted from either mill A or B). Neither cleaning corn nor pellet mill type affected (P > 0.19) nursery pig growth performance. Pelleting improved (P < 0.0001) gain to feed ratio (G:F) by 7.6% compared to mash diets. These data suggest that cleaning is an effective method to legally reduce aflatoxin and fumonisin concentration, but does not impact animal growth performance. Screenings should be used cautiously when feeding to animals.

Evaluating the distribution of African swine fever virus within a feed mill environment following manufacture of inoculated feed.

It is critical to understand the role feed manufacturing may have regarding potential African swine fever virus (ASFV) transmission, especially given the evidence that feed and/or ingredients may be potential vectors. The objective of the study was to evaluate the distribution of ASFV in a feed mill following manufacture of contaminated feed. To accomplish this, a pilot-scale feed mill consisting of a mixer, bucket elevator, and spouting was constructed in a BSL-3Ag facility. First, a batch of ASFV-free feed was manufactured, followed by a batch of feed that had an ASFV-contaminated ingredient added to feed, which was then mixed and discharged from the equipment. Subsequently, four additional ASFV-free batches of feed were manufactured using the same equipment. Environmental swabs from 18 locations within the BSL-3Ag room were collected after each batch of feed was discharged. The locations of the swabs were categorized into four zones: 1) feed contact surface, 2) non-feed contact surface < 1 meter away from feed, 3) non-feed contact surface > 1 meter from feed, and 4) transient surfaces. Environmental swabs were analyzed using a qPCR specific for the ASFV p72 gene and reported as genomic copy number (CN)/mL of environmental swab processing buffer. Genomic copies were transformed with a log10 function for statistical analysis. There was no evidence of a zone × batch interaction for log10 genomic CN/mL (P = 0.625) or cycle threshold (Ct) value (P = 0.608). Sampling zone impacted the log10 p72 genomic CN/mL (P < 0.0001) and Ct values (P < 0.0001), with a greater amount of viral genome detected on transient surfaces compared to other surfaces (P < 0.05). This study illustrates that once ASFV enters the feed mill environment it becomes widespread and movement of people can significantly contribute to the spread of ASFV in a feed mill environment.

Can the digestibility of corn distillers dried grains with solubles fed to pigs at two stages of growth be enhanced through management of particle size using a hammermill or a roller mill?

The objective of this study was to determine the impact of reducing the mean particle size (PS) of corn distillers dried grains with solubles (DDGS) with a hammermill (HM) or with a roller mill (RM) on the apparent total tract digestibility (ATTD) of dry matter (DM), gross energy (GE), N, acid hydrolyzed ether extract (AEE), and fiber components in growing and finishing pigs. Twenty-four growing barrows were housed in individual pens and were randomly assigned to a 3 × 2 factorial design (n = 8): three grinding methods [either corn DDGS ground with an HM to a PS of 450 µm; corn DDGS ground with an RM to a PS of 450 µm; and corn DDGS with a PS of 670 µm (not further ground)] and two body weight (BW) periods (growing pigs with an average initial BW of 54.7 ± 0.9 kg, and finishing pigs with an average initial BW of 107.8 ± 1.5 kg BW). Fecal samples were collected for each BW period in the last 3 d of an 11-d feeding period. Titanium dioxide was used as an indigestible marker. Digestibility data were analyzed using the MIXED procedure of SAS. Results showed that finishing pigs tended to have better ATTD of DM than growing pigs (P = 0.09) and had increased ATTD of GE and N than growing pigs (P = 0.03 and P < 0.01, respectively). On the other hand, growing pigs had better ATTD of AEE than finishing pigs (P = 0.01). Pig BW period did not affect the ATTD of neutral detergent fiber (NDF), acid detergent fiber (ADF), and hemicellulose. Reducing the mean PS of corn DDGS with either HM or RM (from 670 to 450 µm) improved the ATTD of DM and GE (P < 0.01 and P < 0.01), tended to improve the ATTD of N (P = 0.08), and improved the ATTD of AEE (P < 0.01). No effect of reducing PS was observed for the ATTD of NDF, ADF, or hemicellulose. There were no differences between HM and RM in any of the ATTD variables tested. In conclusion, reducing PS of corn DDGS from 670 to 450 µm either with an HM or with an RM improved the digestibility of DM, GE, and AEE and modestly improved the digestibility of N in growing and finishing pigs. However, reducing the PS of corn DDGS did not affect the digestibility of fiber components.