A Proteomic View of the Cross-Talk Between Early Intestinal Microbiota and Poultry Immune System.

Proteomics has been used to investigate cross-talk between the intestinal microbiome and host biological processes. In this study, an in ovo technique and a proteomics approach was used to address how early bacterial colonization in the gastrointestinal tract (GIT) could modulate inflammatory and immune responses in young broilers. Embryos at 18 embryogenic days were inoculated with saline (S), 102 CFU of Citrobacter freundii (CF), Citrobacter species (C2), or lactic acid bacteria mixture (L) into the amnion. At 10 days posthatch, ileum samples from 12 birds per treatment were selected for tandem mass spectrometry analysis. Our further findings indicated that treatment-specific influences on early GIT microbiota resulted in different immune responses in mature broilers. Predicted functional analyses revealed activation of inflammation pathways in broilers treated in ovo with L and CF. Exposure to L enhanced functional annotation related to activation, trafficking of immune cells, and skeletal growth based-network, while CF inhibited biological functions associated with immune cell migration and inflammatory response. These results highlighted that proper immune function was dependent on specific GIT microbiota profiles, in which early-life exposure to L-based probiotic may have modulated the immune functions, whereas neonatal colonization of Enterobacteriaceae strains may have led to immune dysregulation associated with chronic inflammation.

Impact of in ovo administered pioneer colonizers on intestinal proteome on day of hatch.

Pioneer colonization of the gastrointestinal tract (GIT) by bacteria is thought to have major influence on neonatal tissue development. Previous studies have shown in ovo inoculation of embryos with saline (S), species of Citrobacter (C, C2), or lactic acid bacteria (L) resulted in an altered microbiome on day of the hatch (DOH). The present study investigated GIT proteomic changes at DOH in relation to different inoculations. Embryos were inoculated in ovo with S or ∼102 cfu of C, C2, or L at 18 embryonic days. On DOH, the GIT was collected, and tissue proteins were extracted for analysis via tandem mass spectrometry. A total of 493 proteins were identified for differential comparison with S at P ≤ 0.10. Different levels were noted in 107, 39, and 78 proteins in C, C2, and L groups, respectively, which were uploaded to Ingenuity Pathway Analysis to determine canonical pathways and biological functions related to these changes. Three members of the cytokine family (interleukin [IL]-1ß, IL6, and Oncostatin M) were predicted to be activated in C2, indicated with Z-score ≥ 1.50, which suggested an overall proinflammatory GIT condition. This was consistent with the activation of the acute-phase response signaling pathway seen exclusively in C2 (Z-score = 2.00, P < 0.01). However, activation (Z-score = 2.00) of IL-13, upregulation of peroxiredoxin-1 and superoxide dismutase 1, in addition to activation of nitric oxide signaling in the cardiovascular system of the L treatment may predict a state of increased antioxidant capacity and decreased inflammatory status. The nuclear factor erythroid 2-related factor 2 (NRF2)-mediated oxidative stress response (Z-score = 2.00, P < 0.01) was predicted to be upregulated in C which suggested that chicks were in an inflammatory state and associated oxidative stress, but the impact of these pathways differed from that of C2. These changes in the proteome suggest that pioneer colonizing microbiota may have a strong impact on pathways associated with GIT immune and cellular development.

Oxidative metabolism and efficiency: the delicate balancing act of mitochondria.

Mitochondria are responsible for roughly 90% of the ATP produced in a cell. A consequence of aerobic metabolism is oxidative stress that results from production of mitochondrial reactive oxygen species (ROS) due to inefficiency of electron transport. Several antioxidant-redox coupled reactions in the mitochondria help minimize oxidative damage in the mitochondria. These redox reactions not only protect mitochondria from oxidative damage but also are important in regulating cellular redox status. Oxidative stress from mitochondrial ROS occurs in broilers in pulmonary hypertension syndrome, heat stress, and in the phenotypic expression of feed efficiency. Low levels of mitochondrial ROS are now recognized to play important roles in signal transduction mechanisms. A topology of ROS production has been reported that indicates that ROS derived from Complex I primarily cause oxidative damage, whereas ROS generated from Complex III are primarily involved in cell signaling. Reverse electron transport, once considered an artifact of in vitro conditions, now plays significant roles in physiological conditions including inflammation, ischemia-reperfusion, muscle differentiation, and energy utilization. Understanding the balancing act that mitochondria play in health and disease will continue to be vital biological component of improving efficiency in animal production.

Identification of mitochondrial hormone receptors in avian muscle cells.

The major objective of this study was to assess the expression of mitochondrial hormone receptors for progesterone (PR), estrogen (ER), glucocorticoid (GR), thyroid (TR), and insulin (IR) in avian muscle cells (quail muscle 7, QM7) and in breast muscle of quail and broilers. Visualization of receptor location in QM7 cells was accomplished by immunofluorescence. QM7 cells were stained with Mito Tracker Deep Red CMX, fixed in methanol, immune stained with anti-PR, -GR, -TR, -IR, and -ER primary antibodies overnight at 4°C, and visualized with Alexa Fluor 488-conjugated secondary antibody. After staining the nucleus with 4',6-diamidino-2-phenylindole, images were obtained by immunofluorescence microscopy. Merged images revealed the presence of all 5 hormone receptors on mitochondria in QM7 cells. Western blot analysis identified; (a) the ß-isoform of the PR, (b) the α-isoform of GR, (c) the α-receptor of TR, (d) the ß-subunit of IR, and (e) the α-isoform of the ER on mitochondria isolated from broiler breast muscle. Similar results were obtained in quail breast muscle mitochondria with the exception that the α-isoform of the GR was not detected. To our knowledge, this is the first report of hormone receptors (PR, TR, GR, IR, and ER) on mitochondria in avian cells. We hypothesize that these receptors could play important roles in regulating mitochondrial function in avian muscle cells.

Enhanced expression of proteins involved in energy production and transfer in breast muscle of pedigree male broilers exhibiting high feed efficiency.

In cells with fluctuating energy demand (e.g., skeletal muscle), a transfer system of proteins across the inner and outer mitochondrial membranes links mitochondrial oxidative phosphorylation to cytosolic phosphorylated creatine (PCr) that serves as a phosphate reservoir for rapid repletion of cytosolic adenosine triphosphate (ATP). Crucial proteins of this energy transfer system include several creatine kinase (CK) isoforms found in the cytosol and mitochondria. In a recent proteomic study (Kong et al., 2016), several components of this system were up-regulated in high feed efficiency (FE) compared to low FE breast muscle; notably adenine nucleotide translocase (ANT), voltage dependent activated channel (VDAC), the brain isoform of creatine kinase (CK-B), and several proteins of the electron transport chain. Reexamination of the original proteomic dataset revealed that the expression of two mitochondrial CK isoforms (CKMT1A and CKMT2) had been detected but were not recognized by the bioinformatics program used by Kong et al. (2016a). The CKMT1A isoform was up-regulated (7.8-fold, P = 0.05) in the high FE phenotype but there was no difference in CKMT2 expression (1.1-fold, P = 0.59). From these findings, we hypothesize that enhanced expression of the energy production and transfer system in breast muscle of the high FE pedigree broiler male could be fundamentally important in the phenotypic expression of feed efficiency.

Effects of In Ovo feeding of dextrin-iodinated casein in broilers: I. Hatch weights and early growth performance.

This study was conducted to determine the effect of in ovo feeding of dextrin (Dext) and iodinated casein (IC) on hatch and early growth in broilers. Three experiments were conducted at a commercial hatchery using a commercial Inovoject™ system with treatments occurring in conjunction with vaccination at transfer from incubator to hatcher units (18.5 to 19 d embryonic development). In all 3 experiments, approximately 15,000 eggs (2,500 eggs per group) were treated and transferred to a single hatcher unit. Treatments in Exp. 1 consisted of buffered saline solution alone (Control, Cont) or a dextrin solution (Dext, 18% maltodextrin, 10% potato starch dextrin) containing zero, 80, 240, 720, or 2,160 µg IC/mL. The results of this initial experiment indicated that broiler chicks at hatch that received 240 and 720 µg IC/mL in Dext were heavier (P < 0.05) compared to the other treatment groups; there were no differences in hatchability between groups. Based on these findings, subsequent studies used treatments of zero, 240, and 480 µg/mL IC in Dext or Cont. In Exp. 2, hatch weights in all treatment groups were higher (P < 0.05) compared to those receiving Cont. In Exp. 3, chicks given Dext alone or 240 and 480 µg/mL in saline weighed less at hatch compared to the other treatment groups. However, chicks provided Dext alone in Exp. 3 had less weight loss after a 24-hour holding period compared to the other groups. All treatment groups exhibited greater weight gain from one to 10 d compared to the Cont group. The results indicate that in ovo feeding of broiler embryos with Dext containing 240 and 480 µg IC/mL may have beneficial effects on broiler hatch weights and early growth rate.

Effect of in ovo feeding of dextrin-iodinated casein in broilers: II. Hatch window and growth performance.

Studies were conducted using a commercial InovojectTM system to determine effects of in ovo feeding of dextrin and iodinated casein (IC) on hatch and posthatch growth in broilers. At ∼18.5 d embryonic development, eggs were treated with 0, 240, or 480 µg IC/mL in saline (Cont, IC240, and IC480) or dextrin (Dext, DextIC240 and DextIC480). The Dext solution consisted of 18% maltodextrin and 10% potato starch dextrin; saline was the vehicle used by the company for in ovo vaccination. The volume for all in ovo treatments was 50 µL/injection. Eggs in Experiment 1 were transferred to a commercial hatcher unit whereas eggs in Experiments 2 and 3 were transferred to a research hatcher unit to assess effects of treatments on timing of hatch. At hatch, chicks were randomly selected and placed in floor pens and grown to 6 wk. In Experiment 1, there were no differences in hatch weights, but broilers provided Dext IC240 in ovo were heavier (P < 0.05) at 6 wk compared to other treatments with the exception of the Dext IC240 group. In Experiment 2, hatch weights were heavier (P < 0.05) in chicks receiving IC240 and DexIC480 treatments compared to Controls. At 6 wk, broilers in all treatments were heavier (P < 0.05) than Cont with the exception of IC480. In Experiment 3, hatch was stimulated by IC240 (in saline), but was delayed by Dext IC240. Serum analysis of ß-hydroxybutyrate (µM/mL), as an indicator of ketone accumulation from fat metabolism of chicks held in chick boxes for 24 h posthatch (to simulate delay in placement after hatch), indicated that chicks in the IC240 group (that hatched earlier) had higher blood ketones compared to chicks that received Dext or DextIC240 in ovo (that hatched later). We conclude dextrin and iodinated casein (240 µg/mL) provided in ovo (∼18.5 d of embryonic development) has the potential to improve chick quality and posthatch body weight by delaying or narrowing hatch window.

Growth performance, feed digestibility, body composition, and feeding behavior of high- and low-residual feed intake fat-tailed lambs under moderate feed restriction.

Two experiments were conducted to evaluate the effect of moderate feed restriction on productivity of lambs classified on the basis of phenotypic expression of residual feed intake (RFI). In Exp. 1, 58 fat-tailed Kurdi ram lambs (32.1 ± 4.2 kg BW) were individually fed, ad libitum, a pelleted diet (35% alfalfa hay and 65% concentrate). Feed intake and ADG were determined for a 6-wk period and 3 feed efficiency measures including RFI, G:F, and partial efficiency of maintenance (PEM) were calculated. The lambs were sorted based on RFI and the 16 highest RFI (RFI ≥ mean + 0.5 SD) and 16 lowest RFI (RFI ≤ mean - 0.5 SD) lambs were subjected to body composition (BC) and DM digestibility (DMD) analysis. Feeding behavior traits (FB) were also evaluated for 24 h using a regular 5-min interval observation method. The high- and low-RFI lambs (14 lambs/RFI group) so classified in Exp. 1 were used in Exp. 2. Half of the lambs in each RFI group were randomly selected to be fed ad libitum or 85% of ad libitum (restricted feeding), which resulted in 4 experimental groups: 1) ad libitum high-RFI, 2) feed restricted high-RFI, 3) ad libitum low-RFI, and 4) feed restricted low-RFI. The lambs were fed the same diet as Exp. 1, and growth efficiency during a 6-wk test period as well as BC, DMD, and FB were also determined in Exp. 2. In Exp. 1, the low-RFI lambs consumed 14% ( < 0.01) less feed than high-RFI lambs. Differences were also observed between high- and low-RFI groups for G:F ( = 0.01), RFI ( < 0.01), and PEM ( < 0.01) in Exp. 1, but no differences were detected between high- and low-RFI lambs for ADG ( = 0.79), DMD ( = 0.42), BC ( > 0.72), and FB ( > 0.24). In Exp.2, the restriction feeding regime negatively affected ADG ( < 0.01) and G:F ( = 0.02) in low-RFI lambs, whereas G:F ( = 0.02) and PEM ( < 0.01) were improved in high-RFI lambs under the feed restriction condition. No effects of feed restriction on DMD ( = 0.87) and BC ( > 0.05) were observed. The lambs fed at the restricted level of intake presented a greater time ( < 0.01) and rate ( = 0.01) of eating than those fed ad libitum. Although bunk visits and feeding events were decreased ( < 0.01) with feed restriction, no interaction ( > 0.05) was detected between RFI phenotype and feeding regime for FB. In summary, feeding high-RFI lambs at 85% of ad libitum level improved G:F with no effect on ADG, whereas growth performance was reduced by feeding low-RFI lambs at 85% of ad libitum. However, these changes in feed efficiency were not related to DMD, BC, or FB.

Cell bioenergetics in Leghorn male hepatoma cells and immortalized chicken liver cells in response to 4-hydroxy 2-nonenal-induced oxidative stress.

The major objectives of this study were to compare cell bioenergetics in 2 avian liver cell lines under control conditions and in response to oxidative stress imposed by 4-hydroxy 2-nonenal (4-HNE). Cells in this study were from a chemically immortalized Leghorn male hepatoma (LMH) cell line and a spontaneously immortalized chicken liver (CELi) cell line. Oxygen consumption rate (OCR) was monitored in specialized microtiter plates using an XF24 Flux Analyzer (Seahorse Bioscience, Billerica, MA). Cell bioenergetics was assessed by sequential additions of oligomycin, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP), and antimycin-A that enables the determination of a) OCR linked to adenosine triphosphate (ATP) synthase activity, b) mitochondrial oxygen reserve capacity, c) proton leak, and d) nonmitochondrial cytochrome c oxidase activity. Under control (unchallenged) conditions, LMH cells exhibited higher basal OCR and higher OCR attributed to each of the bioenergetic components listed above compared with CELi cells. When expressed as a percentage of maximal OCR (following uncoupling with FCCP), LMH cells exhibited higher OCR due to ATP synthase and proton leak activity, but lower mitochondrial oxygen reserve capacity compared with CELi cells; there were no differences in OCR associated with nonmitochondrial cytochrome c oxidase activity. Whereas the LMH cells exhibited robust ATP synthase activity up to 50 µM 4-HNE, CELi cells exhibited a progressive decline in ATP synthase activity with 10, 20, and 30 µM 4-HNE. The CELi cells exhibited higher mitochondrial oxygen reserve capacity compared with LMH cells with 0 and 20 µM 4-HNE but not with 30 µM 4-HNE. Both cell lines exhibited inducible proton leak in response to increasing levels of 4-HNE that was evident with 30 µM 4-HNE for CELi cells and with 40 and 50 µM 4-HNE in LMH cells. The results of these studies demonstrate fundamental differences in cell bioenergetics in 2 avian liver-derived cell lines under control conditions and in response to oxidative challenge due to 4-HNE.

Gene expression in breast muscle associated with feed efficiency in a single male broiler line using a chicken 44K microarray. II. Differentially expressed focus genes.

Global RNA expression in breast muscle obtained from a male broiler line phenotyped for high or low feed efficiency (FE) was investigated using microarray analysis. Microarray procedures and validation were reported previously. By using an overlay function of a software program (Ingenuity Pathway Analysis, IPA) in which canonical pathways are projected onto a set of genes, a subset of 27 differentially expressed focus genes were identified. Focus genes that were upregulated in the high FE phenotype were associated with important signal transduction pathways (Jnk, G-coupled, and retinoic acid) or in sensing cell energy status and stimulating energy production that would likely enhance growth and development of muscle tissue. In contrast, focus genes that were upregulated in the low FE muscle phenotype were associated with cytoskeletal architecture (e.g., actin-myosin filaments), fatty acid oxidation, growth factors, or ones that would likely be induced in response to oxidative stress. The results of this study provide additional information on gene expression and the cellular basis of feed efficiency in broilers.

Relationship between the activity of mitochondrial respiratory chain complexes and feed efficiency in fat-tailed Ghezel lambs.

This study was conducted to investigate relationships between mitochondrial respiratory chain complex activities, feed efficiency, and carcass traits in sheep. A group of Ghezel male lambs sired by a single ram were randomly allotted to individual pens. The lambs were fed ad libitum with a fattening diet containing 30% roughage (corn silage and alfalfa hay) and 70% concentrate for 70 d to individually phenotype each lamb for feed conversion ratio (FCR), adjusted FCR (aFCR), and residual feed intake (RFI). The lambs were then humanely killed and the liver, abdominal fat, pelvic fat, cardiac fat, warm carcass weight, and cold carcass weight, as well as the cross-sectional area of the LM and the fat depth over the 12th rib, were determined. A portion of LM was obtained to determine mitochondrial protein and respiratory chain complex activities (complexes I to V). Statistical analysis was carried out based on lambs exhibiting high and low RFI (n = 8), FCR (n = 8), or aFCR (n = 8) phenotypes. The lambs exhibiting the high-RFI phenotype consumed 110 g more feed daily (P < 0.05) than did the phenotype exhibiting low RFI, with no difference in ADG. Conversely, there was no difference in feed intake between the low- or high-FCR groups, but sheep exhibiting the low-FCR phenotype gained 70 g more (P < 0.05) per day compared with those exhibiting the high-FCR phenotype. It was determined that all 5 respiratory chain complex activities were greater (P < 0.05) in sheep exhibiting the low-RFI phenotype compared with those exhibiting the high-RFI phenotype, with significant (P < 0.001) negative correlation coefficients between RFI and respiratory chain complex activity. When efficiency was assessed using FCR, only activities of respiratory chain complexes III, IV, and V were less (P < 0.05) in the low-FCR phenotype compared with the high-FCR phenotype, and there were no differences (P > 0.1) in respiratory chain complex activities between groups when FCR was adjusted for metabolic BW (aFCR). There were no differences (P > 0.1) in carcass traits among any of the feed efficiency phenotypes. The results suggest that the inclusion of respiratory chain complex activities in breeding programs may be helpful in selecting for sheep exhibiting the low-RFI phenotype.

Vaccination of chickens with recombinant Salmonella expressing M2e and CD154 epitopes increases protection and decreases viral shedding after low pathogenic avian influenza challenge.

Avian influenza (AI) is a significant public health concern and serious economic threat to the commercial poultry industry worldwide. Previous research demonstrates that antibodies against M2e confer protection against influenza challenge. Using the Red recombinase system in combination with overlapping extension PCR, we recently developed several novel attenuated Salmonella Enteritidis strains that express a protective M2e epitope in combination with a potential immune-enhancing CD154 peptide sequence on the Salmonella outer membrane protein lamB. Commercial Leghorn chicks were orally immunized (immunization dose: 10(6) to 10(8) cfu/chick) with saline (negative control) or one of the recombinant Salmonella strains [DeltaaroA M2e-CD154, DeltahtrA M2e-CD154, DeltaaroA-DeltahtrA M2e(4)-CD154] on day of hatch and 21 d posthatch. These candidate vaccine strains were evaluated for their ability to invade, colonize, and persist in tissues and elicit an M2e-specific antibody response. The vaccine candidate strain DeltaaroA M2e-CD154 exhibited significantly greater organ invasion in the liver and spleen at d 7 (P > 0.05); however, no marked differences in colonization of the cecal tonsils were observed. Vaccinated chickens exhibited significantly increased M2e-specific IgG responses, which were further enhanced by simultaneous expression of CD154 (P < 0.05). Virus neutralization assays gave neutralizing indices of 6.6, 6.3, and 6.3 for DeltaaroA M2e-CD154, DeltahtrA M2e-CD154, and DeltaaroA-DeltahtrA M2e(4)-CD154 seven days post booster immunization, respectively, indicating effective neutralization of AI by serum IgG of vaccinated chickens. In a subsequent direct challenge study, specific-pathogen-free Leghorn chicks immunized with DeltaaroA-DeltahtrA M2e(4)-CD154 offered significant protection against direct challenge with low pathogenic AI H7N2, but not highly pathogenic H5N1 AI. Taken together, these data suggest that these Salmonella-vectored vaccines expressing M2e in association with CD154 are effective at protecting chickens against low pathogenic AI.

Association of mitochondrial function and feed efficiency in poultry and livestock species.

As grain prices have increased dramatically in the past year, understanding the fundamental genetic, cellular, and biochemical mechanisms responsible for feed efficiency (FE; g of gain/g of feed) or residual feed intake (RFI; an alternative feed efficiency trait that quantifies interanimal variation in DMI that is unexplained by differences in BW and growth rate) in livestock and poultry is extremely important with respect to maintaining viable meat production practices in the United States. Although breed and diet have long been known to affect mitochondrial function, few studies have investigated differences in mitochondrial function and biochemistry due to interanimal phenotypic differences in FE or RFI (i.e., variation among animals of the same breed and fed the same diet). This paper reviews existing literature on relationships of mitochondrial function and biochemistry with FE and RFI in poultry and livestock. The overall goal of all of this paper is to assist the development of tools (e.g., genetic markers or biomarkers) to aid commercial breeding companies in genetic selection that, in turn, will help maintain viable livestock and poultry industries in the United States and around the world.

Membrane potential and H2O2 production in duodenal mitochondria from broiler chickens (Gallus gallus domesticus) with low and high feed efficiency.

Increased hydrogen peroxide (H2O2) production was observed in duodenal mitochondria obtained from broiler chickens with low feed efficiency (FE). As a decrease in mitochondrial membrane potential (Deltapsi(m)) due to mild uncoupling of oxidative phosphorylation reduces reactive oxygen species production, this study was conducted to evaluate the effect of uncoupling on Deltapsi(m) and H2O2 production in duodenal mitochondria isolated from broilers with low (0.48+/-0.02) and high (0.68+/-0.01) FE. Membrane potential and H2O2 production were measured fluorometrically and in the presence of different levels of an uncoupler, carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP). The Deltapsi(m) was higher (P

Gene expression in breast muscle and duodenum from low and high feed efficient broilers.

This study was conducted to evaluate messenger RNA (mRNA) expression of genes that are involved in energy metabolism and mitochondrial biogenesis: avian adenine nucleotide translocator (avANT), cytochrome oxidase III (COX III), inducible nitric oxide synthase (iNOS), peroxisome proliferator-activated receptor-gamma (PPAR-gamma), avian PPAR-gamma coactivator-1alpha (avPGC-1alpha), and avian uncoupling protein in breast muscle and duodenum of broilers with low and high feed efficiency (FE). Total RNA was extracted from snap-frozen tissues from male broilers with low (0.55 +/- 0.01) and high (0.72 +/- 0.01) FE (n = 8 per group). Total RNA was reverse-transcribed using oligo(dT), random primers, or both followed by real-time reverse transcription-PCR. Protein oxidation, measured as protein carbonyls, was also evaluated in duodenal mucosa. Protein carbonyls were higher in low FE mucosa in tissue homogenate and mitochondrial fraction. The mRNA expression of iNOS and PPAR-gamma in the duodenum was lower in the low FE broilers, with no differences in avANT, COX III, and avPGC-1alpha. In contrast, expression of avANT and COX III mRNA in breast muscle was lower in low FE broilers with no differences in iNOS, PPAR-gamma, and avPGC-1alpha. The avian uncoupling protein in breast muscle was higher in low FE birds (P = 0.068). These results indicate that there are differences in the expression of mRNA encoding for mitochondrial transcription factors and proteins in breast muscle and duodenal tissue between low and high FE birds. The differences that were observed may also reflect inherent metabolic and gene regulation differences between tissues.

Biochemical evaluation of mitochondrial respiratory chain in duodenum of low and high feed efficient broilers.

Increased H2O2 production, indicating higher oxidative stress, and lower mitochondrial function was previously observed in duodenal mitochondria isolated from broilers with low feed efficiency (FE, gain:feed). Thus, experiments were conducted to 1) evaluate the activity of the respiratory chain complexes (complexes I to V) and 2) assess protein oxidation and mitochondrial protein expression in broilers with low and high FE. Duodenal mitochondria were isolated from broiler breeders with low (0.52 +/- 0.01) and high (0.68 +/- 0.01) FE (n = 8/group). Respiratory chain complex activities were measured spectrophotometrically, whereas mitochondrial protein expression and protein oxidation (carbonyls) were assessed with Western blots. The activities of all complexes, except complex IV, were lower in the low FE compared with high FE mitochondria, whereas protein carbonyl levels were higher in low FE mitochondria. Steady-state levels of 6 out of 7 nuclear-encoded respiratory chain subunits [70S(FP), core I, core II, cytochrome c (cyt c)1, iron-sulfur protein (ISP), and ATPase-alpha] were higher, whereas 3 out of 6 mitochondrial-encoded subunits (ND4, ND6-C, and COX II) were lower in the low FE group, suggesting that sensitivity of mitochondrial proteins to H2O2 or oxidation varies. The general reduction in complex activity and differential protein expression concomitant with higher oxidized proteins in low FE mitochondria suggest that oxidative stress could be contributing to the lower mitochondrial function observed in low FE duodenal mitochondria.

Influence of dietary vitamin E on phagocytic functions of macrophages in broilers.

Vitamin E (VE) is known for its antioxidant properties and has been shown to modulate immune system functions in various species. This study examined the influence of different levels of dietary VE (alpha-tocopherol acetate) on phagocytic functions of macrophages (abdominal exudate cells) in broiler chickens at 3, 5, and 7 wk. Birds were fed commercial diets containing 16 (control), 110, or 220 mg of VE/kg of feed. Macrophages were elicited into the abdominal cavity by injecting a 3% Sephadex solution prepared in PBS (G50-50, 1 mL/100 g of BW) 42 h prior to harvest. The percentage of phagocytically active macrophages and the number of SRBC phagocytosed per macrophage for unopsonized and antibody-opsonized SRBC were determined. These aspects of macrophage function were assessed based on 900 macrophages per sample. When unopsonized SRBC were used, dietary VE supplementation above control level did not affect phagocytic function of macrophages at wk 3, 5, or 7. With antibody-opsonized SRBC, the percentage of phagocytically active macrophages and the number of SRBC phagocytosed per macrophage were higher (P = 0.08 and P = 0.01, respectively) in 3-wk-old birds fed 110 and 220 mg of VE/kg of feed compared with age-matched controls. This enhancing effect of VE supplementation on macrophage function was not observed in 5- and 7-wk-old broilers. It appears from this study that supplemental VE enhances Fc-receptor-mediated macrophage phagocytic activity at early stages of broiler growth.

Differential expression of mitochondrial electron transport chain proteins in cardiac tissues of broilers from pulmonary hypertension syndrome-resistant and -susceptible lines.

Pulmonary hypertension syndrome (PHS) is a metabolic disease associated with the rapid growth rate of modern broilers. Broilers susceptible to PHS experience sustained elevation of pulmonary arterial pressure leading to right ventricular hypertrophy and ultimately heart failure. Previous studies have shown that mitochondrial function is defective in broilers with PHS; they use oxygen less efficiently than broilers without PHS. In this study mitochondrial electron transport chain (ETC) protein levels were compared in cardiac tissues from PHS resistant and susceptible line broilers using quantitative immunoblots. Seven of 9 anti-mammalian mitochondrial ETC protein antibodies tested exhibited cross-species reactivity. Six ETC proteins were differentially expressed in the right ventricles of broilers raised under simulated high altitude conditions (2,900 m above sea level). Four ETC proteins were present at higher levels in resistant line birds without PHS than in resistant line birds with PHS or in susceptible line birds with or without PHS. One ETC protein was present at higher levels in broilers without PHS than in broilers with PHS in both lines, and one ETC protein was present at lower levels in susceptible line birds without PHS than in susceptible line birds with PHS or in resistant line birds with or without PHS. Interestingly, differential expression of mitochondrial ETC proteins was not observed in the right ventricles of broilers raised at local altitude (390 m above sea level) nor was it observed in the left ventricles of broilers exposed to simulated high altitude. These results suggest that higher levels of mitochondrial ETC proteins in right ventricle cardiac muscle may be correlated with resistance to PHS in broilers.

Investigation of proton conductance in liver mitochondria of broilers with pulmonary hypertension syndrome.

We previously reported an impaired ability to regulate hepatic mitochondrial state 4 respiration rate in response to sequential additions of adenosine diphosphate in pulmonary hypertension syndrome (PHS). As proton conductance is a major contributor to State 4 respiration, the major goal of this study was to investigate the nature of proton conductance in hepatic mitochondria isolated from broilers with and without PHS. Broilers were placed on floor litter in environmental chambers and exposed to cold temperatures (15 degrees C) from 3 to 7 wk of age to induce PHS. Liver mitochondria were isolated from birds that exhibited PHS (cyanosis, right ventricular weight ratio > 0.30) or from birds that appeared healthy (no cyanosis, right ventricular weight ratio < 0.27). Isolated mitochondria were placed in a chamber equipped with the ability to measure oxygen content and mitochondrial membrane potential. The mitochondrial membrane potential was assessed by an ion sensitive electrode to measure the distribution of methyltriphenylphosphonium across the inner mitochondrial membrane. Proton conductance was assessed by simultaneously measuring State 4 oxygen consumption rate as respiration was progressively inhibited with increasing concentrations of malonate. The addition of cardiolipin, a lipid found in high concentrations in mitochondrial membranes that can alter proton conductance, had no affect on respiration or mitochondrial membrane potential in either group. The relationship of curves depicting State 4 respiration and mitochondrial membrane potential indicates that PHS mitochondria exhibit impaired substrate oxidation and reduced proton conductance relative to controls. These findings provide further characterization of the altered cellular oxygen utilization in broilers with PHS.

Heart and breast muscle mitochondrial dysfunction in pulmonary hypertension syndrome in broilers (Gallus domesticus).

This study was conducted to determine function and defects in electron transport in muscle mitochondria of meat chickens (broilers) with pulmonary hypertension syndrome (PHS). The respiratory control ratio (RCR, indicative of respiratory chain coupling) was higher in the control than in PHS breast and heart muscle mitochondria, but there were no differences in the ADP/O (an index of oxidative phosphorylation). Sequential additions of ADP improved the RCR in the control breast muscle mitochondria and the ADP/O in PHS breast and heart muscle mitochondria. Basal hydrogen peroxide production, (an indicator of electron leak), was higher in PHS breast and heart muscle mitochondria than in controls and differences in electron leak in PHS mitochondria were magnified by inhibiting electron transport at Complex I and III (cyt b(562)). Complex I activity was lower in PHS heart mitochondria but there was no difference in Complex II activity. Thus, compared to controls, PHS mitochondria exhibited site-specific defects in electron transport within Complex I and III that could contribute to lower respiratory chain coupling. Additionally, it appears that healthy broilers may exhibit higher basal levels of electron leak compared to other avian species. Together, these findings provide insight into inefficient cellular use of oxygen that may contribute to the development of PHS in broilers.