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.

Gene expression in breast muscle associated with feed efficiency in a single male broiler line using a chicken 44K oligo microarray. I. Top differentially expressed genes.

Global RNA expression in breast muscle obtained from a male broiler line phenotyped for high or low feed efficiency (FE) was investigated. Pooled RNA samples (n = 6/phenotype) labeled with cyanine 3 or cyanine 5 fluorescent dyes to generate cRNA probes were hybridized on a 4 × 44K chicken oligo microarray. Local polynomial regression normalization was applied to background-corrected red and green intensities with a moderated t-statistic. Corresponding P-values were computed and adjusted for multiple testing by false discovery rate to identify differentially expressed genes. Microarray validation was carried out by comparing findings with quantitative reverse-transcription PCR. A 1.3-fold difference in gene expression was set as a cutoff value, which encompassed 20% (782 of 4,011) of the total number of genes that were differentially expressed between FE phenotypes. Using an online software program (Ingenuity Pathway Analysis), the top 10 upregulated genes identified by Ingenuity Pathway Analysis in the high-FE group were generally associated with anabolic processes. In contrast, 7 of the top 10 downregulated genes in the high-FE phenotype (upregulated in the low-FE phenotype) were associated with muscle fiber development, muscle function, and cytoskeletal organization, with the remaining 3 genes associated with self-recognition or stress-responding genes. The results from this study focusing on only the top differentially expressed genes suggest that the high-FE broiler phenotype is derived from the upregulation of genes associated with anabolic processes as well as a downregulation of genes associated with muscle fiber development, muscle function, cytoskeletal organization, and stress response.

Investigation of mitochondrial protein expression and oxidation in heart muscle in low and high feed efficient male broilers in a single genetic line.

The objective of this study was to assess the expression of mitochondrial proteins and oxidized proteins in heart muscle homogenate obtained from male broilers exhibiting either high or low feed efficiency (G:F) phenotypes. Tissue homogenate was prepared from ventricular tissue obtained from broilers with high (0.80+/-0.01, n=8) and low (0.62+/-0.02, n=8) FE. The levels of specific immunoreactive proteins and oxidized proteins (carbonyls) were determined using Western blot analysis. The expression of 6 electron transport chain proteins [complex II, 70S subunit (CII 70S); iron-sulfur-containing protein (ISP), cytochrome b (Cyt b), cytochrome (Cyt c1) (of complex III); and cytochrome oxidase subunit II (COX II) (of complex IV)] and adenine nucleotide translocator 1 (ANT1) were higher in low feed efficiency heart mitochondria, but 1 protein [NAD subunit 6c (NAD6c) (complex I)] was higher in high feed efficiency birds. Protein carbonyl levels, indicative of oxidized proteins, were higher in heart tissue of low compared with high feed efficiency broilers.

Improved hatchability and posthatch performance in turkey poults receiving a dextrin-iodinated casein solution in ovo.

Two experiments were conducted with a commercial turkey company using a commercial egg injection system to investigate the effect of a dextrin-iodinated casein solution injected in ovo at 25 d of incubation on turkey poult hatchability, hatch weight, and growth (6 or 7 d posthatch). In experiment 1, a total of 3,900 turkey eggs (1,300 per group) were injected at 25 d of incubation with either 200 µL of a control (physiological saline) solution or a dextrin solution (18% maltodextrin and 10% potato starch dextrin) with 75 or 375 µg/mL of iodinated casein (DexIC75 or DexIC375, where Dex and IC refer to dextrin and iodinated casein, respectively). Two hundred poults from each group were neck-tagged, weighed (hatch weight), placed in a commercial turkey house within a single brooder ring, and weighed again (7 d posthatch). In experiment 2, a total of 5,200 eggs (2,600 per group) were injected with the control or DexIC75 solution. A total of 600 poults (300 per group) were neck-tagged and hatch weights were obtained, followed by placement in a single brooder ring in a commercial house and a second weighing (6 d posthatch). Eggs in experiments 1 and 2 were obtained from hen flocks that were 33 and 5 wk into the laying cycle, respectively. In experiment 1, the DexIC75 injection resulted in a 1.8% increase (P = 0.03) in hatch weight. In experiment 2, the DexIC75 treatment resulted in a 2.4% increase in hatchability (P = 0.01), a 4.3% increase in hatch weight (P < 0.001), and a 1.8% increase in 6-d poult weights (P < 0.03) compared with controls. Results of this study indicate that a solution containing dextrin and 75 µg/mL of iodinated casein injected into turkey eggs at 25 d of incubation may be used to improve early poult weights, hatchability, or both in commercial turkey production.

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.

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.

Mitochondrial proton leak kinetics and relationship with feed efficiency within a single genetic line of male broilers.

Studies were conducted to assess proton leak kinetics (proton conductance) in breast muscle mitochondria isolated from broiler breeder males within a single genetic line exhibiting either high (HFE) or low (LFE) feed efficiency. Proton leak kinetics were determined by simultaneously measuring mitochondrial membrane potential and state 2 (resting) respiration rate in breast muscle mitochondria as succinate oxidation was progressively decreased by malonate. Control proton conductance was similar in HFE and LFE mitochondria and decreased to a similar extent in both groups in response to BSA. Although treatment of mitochondria with Glu or guanosine diphosphate had no effect, retinal increased and carboxyatractylate alone or in combination with Glu decreased proton conductance relative to control proton conductance in both HFE and LFE mitochondria. After treatment with either guanosine diphosphate or carboxyatractylate alone, proton conductance was lower in HFE compared with LFE mitochondria. With the exception of BSA, proton conductance in HFE mitochondria after the various chemical treatments was either less than or equal to, and never greater than, proton conductance in the LFE mitochondria. The results suggest that there are subtle differences in membrane characteristics (e.g., lipids, integral membrane proteins) that affect proton conductance in broiler muscle mitochondria that may in turn play a role in the phenotypic expression of feed efficiency.

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.

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.

Polymorphisms in uncoupling protein, melanocortin 3 receptor, melanocortin 4 receptor, and pro-opiomelanocortin genes and association with production traits in a commercial broiler line.

Because avian uncoupling protein (avUCP), melanocortin 3 receptor (MC3R), melanocortin 4 receptor (MC4R), and pro-opiomelanocortin (POMC) genes may be associated with production traits [e.g., BW, weight gain (WG), and feed conversion ratio (FCR)], male and female broilers from an elite broiler line were screened for polymorphisms in these genes. The PCR-restriction fragment length polymorphism (RFLP) tests were developed to type the missense polymorphisms UCPAla118Val, MC4RSer76Leu, MC3R-Met54Leu, and Gly104Ser and POMCPro61Leu. Of 39 single nucleotide polymorphisms identified in all 4 genes, 24/39 were transitions with 11 having a C to T change. Of the 23 polymorphisms in UCP, 17 represented at least 7 haplotypes in this pedigreed broiler line. The UCP Ala-118Val allele was associated with a) high feed efficiency (FE; P = 0.03) and WG (P = 0.053) in selected males, and b) high BW in selected females (P = 0.07) and unselected males (P = 0.015). The UCPVal118Val allele was found in approximately 10% of the birds that were screened. Five silent substitutions, 3 in MC3R and 2 in MC4R, were also identified. Thirteen polymorphisms were identified in the POMC gene representing at least 3 different alleles. A missense Pro61Leu heterozygote was associated with greater BW in females. The heterozygote MC3R Gly104Ser polymorphism was associated with greater FE in selected males (P = 0.03) and greater BW in unselected males (P = 0.007). The MC4R Ser76Leu heterozygote polymorphism was associated with greater BW than the Leu76 homozygote in females (P = 0.05). From these findings, we hypothesize that UCP, MC3R, MC4R and POMC genes may play important roles and could be candidate loci for production traits such as feed conversion and BW in commercial broiler breeding stock.

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.

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.

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.

Surface wetting strategy prevents acute heat exposure-induced alterations of hypothalamic stress- and metabolic-related genes in broiler chickens.

Heat stress (HS) is devastating to poultry production worldwide, yet its biology and molecular responses are not well defined. Although advances in management strategy have partially alleviated the negative impact of HS, productivity still continues to decline when the ambient temperature rises. Therefore, identifying mechanism-based approaches to decrease HS susceptibility while improving production traits is critical. Recently, we made a breakthrough by applying a surface wetting strategy and showing that it improves growth performance compared with the current conventional cooling system. In the present study, we aimed to further define molecular mechanisms associated with surface wetting in ameliorating HS productivity loss in broilers. Five-week-old broiler chickens were exposed to acute HS (35°C for 2 h) alone or in combination with surface wetting. A control group was maintained at thermoneutral conditions (25°C). Core body temperature (BT) and feed intake were recorded. Blood was collected and hypothalamic tissues (main site involved in the regulation of energy homeostasis) were harvested to determine the expression profile of stress- and metabolic-related genes. Surface wetting prevents HS from increasing BT and plasma corticosterone levels ( < 0.05) and improves feeding and drinking behaviors. At molecular levels, surface wetting blocks the activation of hypothalamic heat shock protein and adenosine monophosphate-activated protein-induced by HS and significantly modulates the expression of feeding-related hypothalamic neuropeptides (agouti-related protein, proopiomelanocortin, orexin, orexin receptor, and leptin receptor). Taken together, our data represent the first evidence that surface wetting alleviates systemic and intracellular stress induced by HS and preserves the intracellular energy status, which, in turn, may result in improved broiler well-being and growth performance.

Feed efficiency and mitochondrial function.

Studies have been conducted in our laboratory to assess differences in mitochondrial function and biochemistry in male broilers with high and low feed efficiency (FE) from the same genetic line and fed the same diet. Mitochondria obtained from broilers with low FE exhibited greater uncoupling of the electron transport chain (ETC) that was apparently due to site-specific defects in electron transport resulting in higher amounts of reactive oxygen species (ROS) compared with high FE mitochondria. Higher amounts of ROS production in Low FE mitochondria were likely responsible for higher protein carbonyl levels, indicative of higher protein oxidation compared with High FE mitochondria and tissue. In turn, higher protein damage in Low FE mitochondria may have contributed to lower activity of electron transport chain complexes relative to values observed in high FE mitochondria. Low FE mitochondria did not exhibit a compromised ability to carryout oxidative phosphorylation, and although there were differences in expression of certain electron transport chain proteins, there was nothing that would indicate that differences in coupling and respiratory chain activity could be due to a general decrease in protein expression between low and high FE mitochondria. The results of these studies provide insight into understanding cellular mechanisms associated with the phenotypic expression of feed efficiency in broilers.

Differential expression of mitochondrial and extramitochondrial proteins in lymphocytes of male broilers with low and high feed efficiency.

Studies were conducted to investigate relationships between mitochondrial and extramitochondrial protein expression, and protein oxidation in lymphocytes obtained from broilers in which individual feed efficiencies were obtained. Lymphocytes were isolated from male broilers from a single line that were shown to exhibit either low (0.48 +/- 0.02, n = 8) or high (0.68 +/- 0.01, n = 7) feed efficiency (FE). Western blot analysis showed that, compared with lymphocytes from high FE broilers, lymphocytes from low FE broilers exhibited a) higher amounts of oxidized proteins (protein carbonyls), b) lower amounts of 3 mitochondrial proteins [core I, cyt c 1 (complex III), and ATP synthase (complex V)], and c) higher amounts of 2 proteins [30 S (complex II) and COX II (complex IV)]. Two-dimensional gel electrophoresis revealed that the intensities of 25 protein spots from pooled samples of lymphocytes from high and low FE broilers differed by 5-fold or more. Three of these protein spots were picked from the gel and subjected to matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry analysis. One protein spot of ~33 kDa was tentatively identified by MALDI-TOF as a fragment of collapsin-2, a component of semaphorin 3D. The results of this study provide further evidence of increased oxidation associated with low FE and further evidence of differential protein expression associated with the phenotypic expression of feed efficiency.

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.