Standardization of a silver stain to reveal mesoscale myelin in histological preparations of the mammalian brain.

BACKGROUND: The brain is built of neurons supported by myelin, a fatty substance that improves cellular communication. Noninvasive magnetic resonance imaging (MRI) is now able to measure brain structure like myelin and requires histological validation. NEW METHOD: Here we present work in small and large biomedical model mammals to standardize a silver impregnation method as a high-throughput histological myelin visualization procedure. Specifically, we built a new staining well plate to increase batch size, and then systematically varied the staining and clearing cycles to describe the staining response curve across taxa and conditions. We compared tissues fixed by immersion or perfusion, mounted versus free-floating, and cut as thicker or thinner slices, with two-weeks of post-fixation. RESULTS: The staining response curves show optimal staining with a single exposure across taxa when incubation and clearing epochs are held to within 3-9 min. We show that clearing was slower in mounted vs free-floating tissue, and that staining was faster and caused fracturing earlier in thinner sliced and smaller volumes of tissue. COMPARISON WITH EXISTING METHODS: We developed a batch processing approach to increase throughput while ensuring reproducibility and demonstrate the optimal conditions for fine myelinated fiber morphology visualization with short cycles (<9 minutes). CONCLUSIONS: We present our optimized protocol to reveal mesoscale neuroanatomical myelin content in histology across mammals. This standard staining procedure will facilitate multiscale analyses of myelin content across development as well as in the presence of injury or disease.

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.

Effects of phytase supplementation and increased nutrient density on growth performance, carcass characteristics, and hypothalamic appetitive hormone expression and catecholamine concentrations in broilers from 1 to 43 days of age.

Two experiments were conducted to evaluate extra-phosphoric effects of phytase and nutrient density on growth performance, meat yield, and hypothalamic appetitive hormone expression and catecholamine concentrations of broilers. Experiment 1 determined differences of digestible amino acid concentrations and AMEn using 256 Yield Plus × Ross 708 broilers (32 cages, 8 birds/cage) fed diets without or with 4,500 phytase units (FTU)/kg inclusion (16 reps/treatment). In Experiment 2, 832 Yield Plus × Ross 708 broilers (32 pens; 26 birds/pen) were provided diets in a 2 × 2 factorial arrangement consisting of 2 nutrient contents (without or with increased density) and 2 phytase inclusions (0 or 4,500 FTU/kg). Increased nutrient density was formulated to contain 0.007, 0.015, 0.013, 0.021, 0.024%, and 61 kcal/kg higher digestible SAA, Lys, Thr, Val, Ile, and AMEn (from Experiment 1) respectively, compared with the control diet. Growth performance was determined at 14, 28, and 40 d of age and carcass characteristics at 41 d of age. At 43 d of age, plasma inositol, hypothalamic appetitive hormone expression, and catecholamine concentrations were determined from 4 birds/pen. Additive effects of phytase inclusion and increased nutrient density resulted in the lowest (P < 0.05) feed conversion from 1 to 40 d of age and the heaviest (P < 0.01) breast meat weights among dietary treatments. Phytase addition numerically increased feed intake (P = 0.06) and BW gain (P = 0.051) compared with birds fed diets without phytase from 1 to 40 d of age. Plasma inositol and dopamine concentrations were 2.3- and 1.2-fold higher (P < 0.01), respectively, in broilers fed phytase-added diets than birds fed diets without phytase inclusion. However, mRNA expression of neuropeptide Y, agouti-related peptide, proopiomelanocortin, cholecystokinin A receptor, ghrelin, and serotonin concentration were not different (P > 0.05) among treatments. These data indicated additive effects of phytase supplementation and increased nutrient density on growth performance and meat accretion of broilers. However, the influence of phytase on feed intake warrants future research.

Sodium buffered formic acid concentration and feed pH is stable over a 3-month period.

Promoting feed hygiene with organic acids is an effective method to prevent foodborne illnesses from bacterial infection. The stability and acidification of mash and pelleted feed with sodium buffered formic acid was investigated. The acid product was incorporated to reach total formate inclusion levels of 0, 6, or 12 g/kg for swine nursery feed; 0, 4, or 9 g/kg for swine finishing feed; and 0, 3, or 6 g/kg for broiler grower feed. Samples were analyzed for total formate and pH on d 4, 32, 60, or 88 post-manufacturing. The concentration of formate remained stable across an 88-d period (P < 0.01). Treatment with the formic acid product decreased feed pH with increasing inclusion levels (all P < 0.01). Within each inclusion level of acid and across time, pH tended to increase in pelleted feed and decrease in mash feeds (all P < 0.01); however, these changes were small (0.1 units pH). These data suggest that sodium buffered formic acid can be applied to both mash and pelleted feed to provide continuous acidification over a 3-month period.

Dietary supplementation with anti-IL-10 antibody during a severe Eimeria challenge in broiler chickens.

Attenuation of host IL-10 activity during Eimeria infection may elicit a robust Th1 response to eliminate the parasite from the gut epithelium. An experiment was conducted to study the effects of feeding IL-10 neutralizing antibody delivered via a dried egg product (DEP) on growth performance, immune responsivity, and gut health outcomes during a severe challenge with either Eimeria acervulina (study 1) or Eimeria tenella (study 2) following FDA CVM #217 protocol to test anticoccidial products. A total of 720 male Ross 308 chicks were used in each study, with 15 replicate cages of 12 birds and the following 4 treatments: sham-inoculated (uninfected) control diet (UCON), Eimeria-infected control diet (ICON), and Eimeria-infected control diet supplemented with DEP at 2 levels (165 [I-165] or 287 [I-287] U/tonne in study 1 and 143 [I-143] or 287 [I-287] U/tonne in study 2). Individual birds assigned to infected treatment groups received a single oral dose of either 200,000 E. acervulina (study 1) or 80,000 E. tenella (study 2) oocysts at 12 d of age (i.e., d post inoculation [DPI] 0), whereas uninfected birds were sham-inoculated with tap water. A one-way ANOVA was performed on outcomes including growth performance, hematology, serum chemistry profiles, immunophenotyping profiles, and intestinal lesion scores. In both studies, DPI 0 to 7 weight gain, feed intake, and feed conversion ratio were worse (P < 0.05) in all infected groups compared with the UCON group. Compared with ICON, DEP supplementation elicited no differences on overall growth performance. Histopathology and lesion scores revealed severe damage to the gut epithelium owing to the Eimeria challenge, yet DEP supplementation did not improve these outcomes or oocyst shedding, hematological measurements, or serum chemistry. However, DEP supplementation improved (P < 0.05) the percentage of circulating CD3+ cells at 6 DPI in study 2. These results indicate that DEP does not appear to elicit a coccidiostatic effect during a severe infection with E. acervulina or E. tenella.

Effects of methylsulfonylmethane and neutralizing anti-IL-10 antibody supplementation during a mild Eimeria challenge infection in broiler chickens.

A 28-day experiment was conducted in broilers to study the effects of feeding methylsulfonylmethane (MSM) and IL-10-neutralizing antibody from dried egg product (DEP) on the growth performance, immune responsivity, oxidative stress parameters, and gut health outcomes during a mild infection with mixed species of Eimeria. A total of 500 male Ross 308 chicks were allocated to five treatments: sham-inoculated (uninfected) chickens fed control diet (UCON), Eimeria-infected chickens fed control diet (ICON), and Eimeria-infected chickens fed control diet supplemented with 287 U/tonne of DEP (I-DEP), 0.4% MSM, or their combination (I-DEP-MSM), with 10 replicate cages of 10 birds per treatment. All infected groups received 1 mL of an oral inoculum containing Eimeria acervulina (10,000 oocysts), Eimeria maxima (5,000 oocysts), and Eimeria tenella (5,000 oocysts) on study days 7 and 14. Data were analyzed as a two-way ANOVA for all treatments including Eimeria-infected groups, in addition to a single degree of freedom contrast to compare uninfected and infected groups receiving the control diet. Mild Eimeria infection did not influence the growth performance in ICON compared with UCON at any time points. Overall (day 0-28) growth performance parameters were not influenced by either infection or dietary supplementation of MSM or DEP. However, birds in I-DEP-MSM showed improved ADG during study day 7 to 14 (i.e., 7 d after primary inoculation) indicating a beneficial effect immediately after Eimeria infection. Although MSM supplementation reduced thiobarbituric acid reactive substances (day 21 and 28), both MSM and DEP improved the total antioxidant capacity (day 21) in the plasma of infected birds. Histopathological outcomes were not influenced by treatments, and fecal oocyst output was higher in MSM- and DEP-supplemented groups than with ICON, indicating no beneficial effects. Similarly, expression of cecal inflammatory cytokines (IL-10, IL-1ß, and interferon-γ) was not affected by MSM, DEP, or their combination. Overall, the current results suggest that both MSM and DEP supplementation may benefit birds during a mild Eimeria infection as indicated by improvements in ADG and oxidative stress outcomes.

Dietary pectin at 0.2% in milk replacer did not inhibit growth, feed intake, or nutrient digestibility in a 3-week neonatal pig study.

Pectin is a fermentable soluble fiber that can be used as a thickener in formulas for infants and young children. The Joint FAO/WHO Expert Committee on Food Additives concluded that pectin is not of concern for inclusion in infant formula at up to 0.2%. As part of the safety assessment of the suitability of pectin for young infants (≤12 weeks of age), we conducted a 3-week dietary study in a neonatal pig model to 1) investigate the impact of pectin at different doses on neonatal pigs' growth and 2) explore the potential explanation for the dose response. Male and female neonatal pigs were fed milk replacer containing 0, 0.2%, or 1% pectin beginning on postnatal day 2 for 21 days. Body weight, feed intake, and apparent ileal digestibility of dry matter, crude protein, and energy were reduced in pigs fed diets containing 1% pectin (P < 0.01) but not in pigs fed with 0.2% pectin. These data indicate that inclusion of pectin in the diet at 0.2%, equivalent to 704 mg/kg BW/day is safe, well tolerated, and did not result in any adverse health effects in this neonatal pig study.

Dietary methylsulfonylmethane supplementation and oxidative stress in broiler chickens.

Methylsulfonylmethane (MSM) is an organic, sulfur-containing compound widely used as a dietary supplement to improve joint health and treat arthritic pain. An experiment was conducted to study the effects of feeding 0.05% MSM to broilers exposed to diet-induced oxidative stress on tissue MSM distribution, growth performance, oxidative stress biomarkers, and immune responsivity. A total of 528 birds were allocated to 4 dietary treatments (fresh oil-no MSM, fresh oil-MSM, oxidized oil-no MSM, oxidized oil-MSM) as provided ad libitum to 11 replicate cages of 12 birds per treatment. Blood and tissue samples were collected to analyze MSM concentrations, and oxidative stress biomarkers including concentrations of thiobarbituric acid reactive substances (TBARS), total antioxidant capacity (TAC), total glutathione, and glutathione peroxidase (GPx) and reductase (GR) activities. Additionally, blood samples collected at day 25 were used to quantify T-cell (TC) populations using flow cytometry. Overall, MSM was quantified in all tissues and plasma samples of MSM-treated groups at all time points. Oxidized oil reduced (P = 0.006) feed intake over the 21-d feeding period, but MSM did not affect growth equally across time points. No effects (P > 0.2) of MSM or oil type were observed on TC populations. In the presence of oxidized oil, MSM reduced (P = 0.013) plasma TBARS and increased (P = 0.02) liver GPx at day 21, and increased (P = 0.06) liver GR at day 7. Irrespective of dietary oil type, groups supplemented with MSM showed higher plasma TAC at day 7 (P = 0.023), liver GPx activity at day 21 (P = 0.003), and liver GR activity at day 7 (P = 0.004) compared with groups not receiving MSM. In conclusion, 0.05% dietary MSM supplementation partially protected birds from oxidative stress but did not affect immune cell profiles.

Toxicity and tissue distribution of methylsulfonylmethane following oral gavage in broilers.

An experiment was conducted to investigate the toxicity and tissue distribution of methylsulfonylmethane (MSM) following oral gavage in broilers. A total of four hundred and thirty-two 15-day-old Ross 308 male broilers were allotted to 6 treatments with 6 replicates of 12 birds per replicate and administered a single oral dose of MSM at 0, 50, 100, 300, 1,000, or 2,000 mg/kg BW (Study 1). Another one hundred and sixty-eight 3-day-old chicks were allotted to either control or test group (Study 2) and administered a daily oral gavage of either 0 or 1, 500 mg/kg BW of MSM for 21 D consecutively. Blood and tissue samples were collected over a 48 h (Study 1) or 21 D (Study 2) period and analyzed for MSM concentrations. Toxicity was assessed through changes in hematology and clinical blood chemistry. In Study 1, plasma MSM concentrations were below 167 µg/mL at all time-points in birds receiving up to 300 mg/kg BW, and were significantly higher (P < 0.05) in birds receiving 1,000 or 2,000 mg/kg BW. Similarly, only the highest 2 MSM dosages elicited increased lymphocyte and decreased heterophil counts at 8 h (P < 0.003) and decreased hematocrit at 48 h (P = 0.015). Growth performance variables were unaffected by MSM in Study 2, and plasma and tissue MSM concentrations were highest on study day 21, with MSM-dosed birds always exhibiting higher (P < 0.03) concentrations compared with the control. Birds in Study 2 that were dosed with MSM had decreased liver enzyme concentrations at day 7 and 21 and decreased glucose and phosphorus at day 7. Importantly, MSM was detected in plasma and all tissues of control groups, confirming that MSM is synthesized de novo in chickens. In conclusion, oral MSM at either acute (single dose at 1,000 to 2,000 mg/kg BW) or sub-chronic (1,500 mg/kg BW daily for 21 D) concentrations did not cause any adverse effects on growth or clinical outcomes and appeared to be absorbed and distributed throughout the body.

Guanidinoacetic acid is efficacious in improving growth performance and muscle energy homeostasis in broiler chicks fed arginine-deficient or arginine-adequate diets.

Two studies were conducted to test the efficacy of guanidinoacetic acid (GAA) to spare Arg and serve as a precursor of creatine (Cr) by evaluating growth performance and muscle cellular energy homeostasis in broiler chicks. In both studies, 12 replicate pens of 6 chicks received dietary treatments beginning at day 2 post-hatch. At conclusion of each study, muscle biopsy samples were collected within 60 s of euthanasia for analysis of Cr-related energy metabolites. In study 1, Arg-deficient starter and grower basal diets were supplemented with 0 (negative control, NC), 0.06, 0.12, or 0.18% GAA, or supplemental Arg (positive control, PC; 0.37 and 0.32% L-Arg in starter and grower phases, respectively). Dietary GAA elicited graded improvements, with final BW, overall BW gain, and overall G:F being increased (P < 0.05) by 0.12% GAA compared with the NC diet with no difference to PC diet. Increases (P < 0.001) of phosphocreatine (PCr), total Cr (tCr), and glycogen concentrations, as well as the PCr-to-adenosine triphosphate (ATP) and glycogen:ATP ratios, were observed with supplementation of 0.12% GAA compared with the NC diet, even exceeding responses to the PC diet. In study 2, Arg-adequate starter and grower basal diets were supplemented with 0 (negative control, NC), 0.06, or 0.12% GAA, 0.12% Cr monohydrate (PC1), or salmon protein (PC2; containing total Arg concentrations equal to those of the NC diet in each phase and containing similar Cr as in PC1). Overall G:F was increased (P < 0.05) by PC1, but not by PC2, compared with the NC, while GAA supplementation elicited a response intermediate to NC and PC1 diets. However, GAA supplementation increased (P < 0.01) concentrations of tCr and glycogen, as well as the PCr:ATP and glycogen:ATP ratios, when compared with the NC (Arg-adequate) diet. Collectively, these data indicate that GAA can be used to replace Arg in practical, Arg-deficient diets and improve muscle energy homeostasis in broiler chicks receiving either Arg-deficient or Arg-adequate practical diets.

Effects of Yucca schidigera-derived saponin supplementation during a mixed Eimeria challenge in broilers.

An experiment was conducted to determine if dietary Yucca-derived saponin supplementation could ameliorate the immune and growth responses of broilers during a mixed coccidian challenge. A total of 576 two-day-old male Ross 308 broiler chicks were housed in galvanized starter batteries and randomly assigned to 1 of 4 dietary treatment groups (12 replicate cages of 12 birds). Dietary treatments were corn-soybean meal-based and included 1) control diet + sham-inoculated (Ucon), 2) control diet + Eimeria oocyst challenge (Icon), 3) control diet with 250 mg/kg Yucca-derived saponin product + Eimeria oocyst challenge (ISap250), and 4) control diet with 500 mg/kg of Yucca-derived saponin product + Eimeria oocyst challenge (ISap500). On study day 14, birds were orally inoculated with 1.5 mL of tap water containing Eimeria acervulina, E. maxima, and E. tenella (100,000, 40,000, and 30,000 oocysts/dose, respectively), or sham-inoculated with 1.5 mL of tap water. Eimeria-challenged birds exhibited a reduction in growth compared with uninfected birds (P < 0.001); however, there were no detectable differences due to dietary treatment among Eimeria-challenged groups. Mucosal thickness in the jejunum was increased (P < 0.042) in all infected groups and there were no differences among infected groups; however, saponin supplementation included at 250 mg/kg was not significantly different from the uninfected birds. Lymphocytes as a percentage of total white blood cells were increased (P < 0.014) in all Eimeria-challenged groups at 7 D post-inoculation compared with uninfected birds, but birds supplemented at 250 mg/kg were not different from uninfected birds. Cecal and duodenal IFN-γ expression increased with infection when compared with sham-inoculated birds. Cecal and duodenal IL-1ß expression increased (P < 0.008 and P < 0.039) due to infection, and ISap250 and ISap500 treatments ameliorated IL-1ß expression to levels not different from sham-inoculated birds. These results suggest that saponin supplementation may provide some immunomodulatory effects during a mixed coccidian challenge as evidenced by lymphocyte responses, changes in intestinal structure, and alterations in cecal and duodenal inflammatory cytokine mRNA expression.

Influence of feeding thermally peroxidized soybean oil on oxidative status in growing pigs.

The objectives of this study were to determine whether feeding thermally processed peroxidized soybean oil (SO) induces markers of oxidative stress and alters antioxidant status in pig tissue, blood, and urine. Fifty-six barrows (25.3 ± 3.3 kg initial BW) were randomly assigned to dietary treatments containing 10% fresh SO (22.5 °C) or thermally processed SO (45 °C for 288 h, 90 °C for 72 h, or 180 °C for 6 h), each with constant air infusion rate of 15 liters/minute. Multiple indices of lipid peroxidation were measured in the SO including peroxide value (2.0, 96, 145, and 4.0 mEq/kg for 22.5, 45, 90, and 180 °C processed SO, respectively) and p-anisidine value (1.2, 8.4, 261, and 174 for 22.5, 45, 90, and 180 °C processed SO, respectively); along with a multitude of aldehydes. Pigs were individually housed and fed ad libitum for 49 d which included a 5 d period in metabolism crates for the collection of urine and serum for measures of oxidative stress. On day 49, pigs were euthanized to determine liver weight and analyze liver-based oxidative stress markers. Oxidative stress markers included serum, urinary, and liver thiobarbituric acid reactive substances (TBARS), and urinary F2-isoprostanes (ISP) as markers of lipid damage; serum and liver protein carbonyls (PC) as a marker of protein damage; and urinary and liver 8-hydroxy-2'-deoxyguanosine (8-OH-2dG) as a marker of DNA damage. Superoxide dismutase (SOD), and catalase activity (CAT) were measured in liver, glutathione peroxidase activity (GPx) was measured in serum and liver, and ferric reducing antioxidant power (FRAP) was measured in serum and urine as determinants of antioxidant status. Pigs fed 90 °C SO had greater urinary ISP (P = 0.02), while pigs fed the 45 °C SO had elevated urinary TBARS (P = 0.02) in comparison to other treatment groups. Pigs fed 45 °C and 90 °C SO had increased serum PC concentrations (P = 0.01) and pigs fed 90 °C SO had greater (P = 0.01) liver concentration of 8-OH-2dG compared to pigs fed the other SO treatments. Furthermore, pigs fed 90 °C SO had reduced serum GPx activity in comparison to pigs fed fresh SO (P = 0.01). In addition, pigs fed 180 °C SO had increased liver CAT activity (P = 0.01). Liver GPx and SOD or serum and urinary FRAP were not affected by dietary treatment. These results indicate that dietary peroxidized soybean oil induced oxidative stress by increasing serum PC while diminishing serum GPx, increasing urinary ISP and TBARS, and increasing 8-OH-2dG and CAT in liver.

Efficacy of guanidinoacetic acid on growth and muscle energy metabolism in broiler chicks receiving arginine-deficient diets.

Guanidinoacetic acid (GAA) is formed from arginine (Arg) and is the immediate precursor to creatine (Cr) and phosphocreatine (PCr), which are important compounds involved in muscle energy homeostasis. This study sought to determine whether GAA could spare Arg in broiler chicks fed an Arg-deficient practical diet. A basal [0.84% standardized ileal digestible (SID) Arg] was supplemented with combinations of L-Arg (0 or 0.16%) and GAA (0, 0.06, or 0.12%) to form a factorial arrangement of diets; the unsupplemented basal served as the negative control (NC). Additionally, the basal was supplemented with 0.32% Arg to generate an Arg-adequate positive control (PC). Mash diets were fed to 8 replicate pens of 5 chicks per treatment from d 8 to 22 posthatch, with measurements including growth performance, blood GAA metabolites, muscle cellular energy markers, and clinical outcomes. Supplementation of 0.16% Arg increased (P < 0.05) BW gain from d 15 to 22 posthatch, while graded addition of GAA tended to improve BW gain (P < 0.094). Supplementation of either Arg or GAA increased (P < 0.05) feed efficiency from d 15 to 22 and d 8 to 22 posthatch. Birds fed the PC diet had greater (P < 0.05) responses for nearly all blood and tissue outcomes compared with NC-fed birds. Serum GAA was more responsive to supplementation of GAA in the presence versus absence of supplemental Arg (interaction, P < 0.001). Interactions (P < 0.05) were also observed for concentrations of muscle total Cr, creatinine, and most serum essential amino acids, notably Arg. Serum Cr, as well as muscle PCr, total Cr, and glycogen were increased (P < 0.05) independently by Arg and GAA supplementation, with highest levels achieved via combined addition of 0.12% GAA and 0.16% Arg. Minimal effects were detected on hematological and clinical chemistry outcomes. Overall, we conclude that GAA supplementation can spare Arg in broiler chicks fed Arg-deficient practical diets as evidenced by improvements in growth performance and muscle energy stores.

Effects of a high level of phytase on broiler performance, bone ash, phosphorus utilization, and phytate dephosphorylation to inositol.

An experiment was conducted to evaluate how the addition of microbial phytase influenced growth performance, bone mineralization, tissue P content, apparent digestibility and retention, and inositol phosphate (IP) concentrations in broilers fed diets with varying mineral matrices from 2 to 23 d of age. At 2 d of age, chicks were randomly allotted to receive 1 of 6 experimental diets arranged as a 3 × 2 factorial of mineral matrix [control diet with 1.0% Ca and 0.5% non-phytate phosphorus (NPP); mineral matrix 1 with 0.84% Ca and 0.35% NPP; and mineral matrix 2 with 0.77% Ca and 0.29% NPP] and phytase supplementation (0 or 1,500 FTU/kg). Feed intake was influenced (quadratic, P = 0.012) by the mineral matrix, but no interaction or main effect of phytase were observed. Phytase increased (P = 0.011) BW gain regardless of the mineral matrix applied. Feed efficiency was not influenced (P > 0.05) by mineral matrix, phytase, or their interaction. Phytase increased bone ash content differentially across matrices (interaction, P < 0.01), and tibia P content was lowest in birds fed matrix 2 and highest in the control (linear, P < 0.05). Concentrations of P in muscle, spleen, and liver were not affected by treatment. An interactive effect (P < 0.01) was observed for apparent ileal digestibility (AID) of P, where phytase increased AID in matrix 1. An interactive effect (P < 0.01) was observed for apparent retention of P and Ca, where phytase reduced P and Ca retention in the control diet. A main effect (P < 0.01) of mineral matrix was observed for AID of Ca, with birds fed matrix 1 having the lowest AID of Ca compared with control and matrix 2 treatments. Phytase influenced (P < 0.05) IP concentrations differently across matrices. Overall, phytase and the mineral matrix, either as main effects or in an interactive manner, influenced growth performance, apparent nutrient digestibility and retention, bone and IP concentration responses in broiler chicks.

Influence of dietary calcium concentrations and the calcium-to-non-phytate phosphorus ratio on growth performance, bone characteristics, and digestibility in broilers.

Two experiments were conducted to determine the influence of dietary Ca concentrations (Experiment 1) and a combination of dietary Ca and non-phytate phosphorus (NPP) to create distinct Ca-to-NPP ratios (Experiment 2) in corn-soybean meal diets fed to broiler chickens from 2 to 23 d of age. In Experiment 1, dietary treatments consisted of 7 concentrations of Ca (0.4, 0.6, 0.8, 1.0, 1.2, 1.4, or 1.6% of the diet; 7 treatments total), and NPP concentrations were maintained at 0.3%. Increasing the dietary Ca concentration while maintaining 0.3% NPP elicited linear reductions (P < 0.01) in overall growth performance and tibia ash. Dietary effects also were observed for apparent retention of P and Ca, which decreased (P < 0.05) linearly or quadratically for birds receiving dietary treatments with Ca concentrations greater than 0.6%. In Experiment 2, diets were formulated to contain 3 concentrations of Ca (0.4, 1.0, or 1.6% of the diet) with NPP concentrations either constant at 0.45% or adjusted to maintain a dietary Ca-to-NPP ratio of 2:1 (6 treatments total). Growth performance was not influenced by Ca concentration or the Ca-to-NPP ratio. Tibia break force was lower (P < 0.01) in birds fed diets containing 0.4% Ca, regardless of the NPP concentration. Tibia ash increased (P < 0.01) as the dietary Ca concentration increased. Neither the dietary Ca nor NPP concentrations affected nitrogen retention (P > 0.05). Upon maintaining a constant 2:1 Ca-to-NPP ratio, P and Ca retention decreased (P < 0.01) at the highest Ca concentration. In conclusion, imbalanced Ca and NPP adversely influenced growth performance and nutrient retention of broilers, indicating the concentrations of Ca and NPP required to maximize bone structure and function may be higher than those required for performance.

Effects of dietary copper and amino acid density on growth performance, apparent metabolizable energy, and nutrient digestibility in Eimeria acervulina-challenged broilers.

The objective of this experiment was to evaluate the influence of copper supplementation in diets varying in amino acid (AA) density on growth performance, apparent metabolizable energy (AMEn), apparent ileal nutrient digestibility (AID), and plasma carotenoids in broilers infected with Eimeria acervulina. Ross 308 male broilers (480 total) were housed in battery cages and allotted to 8 experimental treatments in a factorial arrangement of 2 dietary AA densities [1.00% (LAA) or 1.20% (HAA) digestible Lys], 2 supplemental copper concentrations (zero or 116 mg/kg), and 2 E. acervulina infection states (uninfected or infected). Essential AA ratios relative to digestible Lys were similar in both the LAA and HAA diets, and copper was provided by 200 mg/kg of tribasic copper chloride (58% copper). Chicks received experimental diets from 2 to 21 d post hatch and 6 replicate cages of 10 birds per cage were assigned to each treatment. Broilers were inoculated with zero or 6.3 × 105 sporulated E. acervulina oocysts at 15 d and blood and ileal digesta were collected at 21 days. From 2 to 15 d, body weight gain and G:F of broilers were improved (P < 0.05) with increasing AA density, and an AA density × copper interaction was observed (P < 0.05) for feed intake. Eimeria infection reduced (P < 0.05) plasma carotenoids, growth performance, dietary AMEn, and AID of organic matter, nitrogen, and total AA. There were no interactive effects of dietary treatments with E. acervulina infection on broiler growth performance or dietary AMEn. An AA density × copper supplementation interaction was observed (P < 0.05) for AID of total AA, whereby copper supplementation increased AID of total AA for birds fed the LAA diet and decreased AID of total AA for birds fed the HAA diet. In summary, E. acervulina-induced reductions in nutrient digestibility were dependent on dietary copper and AA status, but changes in digestibility had minimal impact on growth performance of broilers during the E. acervulina infection period.

Interactive effects of dietary arginine and Eimeria acervulina infection on broiler growth performance and metabolism.

The influence of dietary Arg concentration and Eimeria acervulina infection on broiler growth performance and plasma carotenoid, nitric oxide (NO), amino acid, and urea concentrations was evaluated. Male Ross × Ross 308 broilers (384 total) were fed a common diet for 10 d post-hatch and provided experimental diets formulated to contain 1.23 (HA) or 0.74% (LA) standardized ileal digestible Arg from 10 to 28 d. At 21 d, one-half of the broilers were switched to the opposite diet to create 4 dietary regimens where birds were fed the LA diet throughout, the LA diet replaced by the HA diet at 21 d, the HA diet throughout, or the HA diet replaced by the LA diet at 21 d. Broilers were orally inoculated 0 (uninfected) or 3.5 × 105 sporulated E. acervulina oocysts at 15 d, resulting in a factorial arrangement of 4 dietary regimens × 2 infection states (8 replicates/treatment). Overall (10 to 28 d) BW gain and G:F were greatest (P < 0.01) for birds fed the HA diet throughout, regardless of infection status. Eimeria acervulina infection decreased (P < 0.01) BW gain of birds from 15 to 21 d, and G:F was lowest (P < 0.05) for infected birds fed the LA diet during this period. There was no influence (P > 0.05) of E. acervulina on BW gain or G:F of broilers from 21 to 28 d. Plasma Arg, Lys, and Orn levels at 21 d indicated that the LA diet caused an imbalance in the Lys and Arg status of broilers, and E. acervulina infection increased (P < 0.01) the plasma concentration of these 3 amino acids. Diet × infection interactions (P < 0.05) were observed on 21 d for plasma carotenoids and NO, whereby infection decreased plasma carotenoids and increased plasma NO, but dietary Arg concentration only influenced these measures for uninfected birds. Thus, production of NO during E. acervulina infection was not impaired by dietary Arg limitation.

Influence of dietary amino acid reductions and Eimeria acervulina infection on growth performance and intestinal cytokine responses of broilers fed low crude protein diets.

Two experiments were conducted to evaluate the influence of Eimeria acervulina infection on growth performance, plasma carotenoids, and intestinal cytokine responses of broilers fed low crude protein (LCP) diets with reduced concentrations of selected amino acids (AA). Experiment 1 was conducted to validate a dietary formulation approach in which broilers were fed 1 of 5 diets including a 19.0% CP corn-soybean meal based (CSBM) diet, a LCP control diet (16.7% CP) that matched the AA profile of the CSBM diet, and 3 LCP diets with 30% reductions in standardized ileal digestible concentrations of TSAA, Lys, or Thr from 14 to 23 d post-hatch. Body weight gain and G:F were greatest (P < 0.05) and similar for broilers fed the CSBM and LCP control diets, whereas reductions in Thr, TSAA, and Lys each decreased (P < 0.05) G:F of broilers. In Experiment 2, birds were allotted to 18 treatment groups in a factorial arrangement of 9 dietary treatments × 2 infection states. Dietary treatments included a LCP control diet similar to that fed in Experiment 1 and 8 LCP diets with 40% individual reductions in TSAA, Lys, Thr, Val, Ile, Arg, Phe + Tyr, or Gly + Ser. Broilers received experimental diets from 10 d to 28 d post-hatch and were inoculated with 0 or 4.0 × 105 sporulated E. acervulina oocysts at 15 d. Decreased (P < 0.05) growth performance (10 to 28 d) of broilers was observed with each AA reduction, except Phe + Tyr, compared with birds fed the LCP control diet. Body weight gain and G:F were lowest (P < 0.05) for birds fed diets reduced in Lys or Val. Eimeria acervulina decreased growth performance and plasma carotenoids of broilers, but effects varied among dietary treatment groups as indicated by diet × infection interactions (P < 0.05). Dietary AA reductions did not alter (P > 0.05) the increase in intestinal gene expression of interferon-γ, interleukin-1ß, or interleukin-10 observed in E. acervulina-infected birds.