Michael E. Persia

RNA-seq analysis of broiler liver transcriptome reveals novel responses to high ambient temperature

BackgroundIn broilers, high ambient temperature can result in reduced feed consumption, digestive inefficiency, impaired metabolism, and even death. The broiler sector of the U.S. poultry industry incurs approximately

Transcriptome Analysis of Post-Hatch Breast Muscle in Legacy and Modern Broiler Chickens Reveals Enrichment of Several Regulators of Myogenic Growth

52 million in heat-related losses annually. The objective of this study is to characterize the effects of cyclic high ambient temperature on the transcriptome of a metabolically active organ, the liver. This study provides novel insight into the effects of high ambient temperature on metabolism in broilers, because it is the first reported RNA-seq study to characterize the effect of heat on the transcriptome of a metabolic-related tissue. This information provides a platform for future investigations to further elucidate physiologic responses to high ambient temperature and seek methods to ameliorate the negative impacts of heat.ResultsTranscriptome sequencing of the livers of 8 broiler males using Illumina HiSeq 2000 technology resulted in 138 million, 100-base pair single end reads, yielding a total of 13.8 gigabases of sequence. Forty genes were differentially expressed at a significance level of P-value < 0.05 and a fold-change ≥ 2 in response to a week of cyclic high ambient temperature with 27 down-regulated and 13 up-regulated genes. Two gene networks were created from the function-based Ingenuity Pathway Analysis (IPA) of the differentially expressed genes: “Cell Signaling” and “Endocrine System Development and Function”. The gene expression differences in the liver transcriptome of the heat-exposed broilers reflected physiological responses to decrease internal temperature, reduce hyperthermia-induced apoptosis, and promote tissue repair. Additionally, the differential gene expression revealed a physiological response to regulate the perturbed cellular calcium levels that can result from high ambient temperature exposure.ConclusionsExposure to cyclic high ambient temperature results in changes at the metabolic, physiologic, and cellular level that can be characterized through RNA-seq analysis of the liver transcriptome of broilers. The findings highlight specific physiologic mechanisms by which broilers reduce the effects of exposure to high ambient temperature. This information provides a foundation for future investigations into the gene networks involved in the broiler stress response and for development of strategies to ameliorate the negative impacts of heat on animal production and welfare.

Effects of Vitamin D3-Enriched Diet on Egg Yolk Vitamin D3 Content and Yolk Quality

Agriculture provides excellent model systems for understanding how selective pressure, as applied by humans, can affect the genomes of plants and animals. One such system is modern poultry breeding in which intensive genetic selection has been applied for meat production in the domesticated chicken. As a result, modern meat-type chickens (broilers) exhibit enhanced growth, especially of the skeletal muscle, relative to their legacy counterparts. Comparative studies of modern and legacy broiler chickens provide an opportunity to identify genes and pathways affected by this human-directed evolution. This study used RNA-seq to compare the transcriptomes of a modern and a legacy broiler line to identify differentially enriched genes in the breast muscle at days 6 and 21 post-hatch. Among the 15,945 genes analyzed, 10,841 were expressed at greater than 0.1 RPKM. At day 6 post-hatch 189 genes, including several regulators of myogenic growth and development, were differentially enriched between the two lines. The transcriptional profiles between lines at day 21 post-hatch identify 193 genes differentially enriched and still include genes associated with myogenic growth. This study identified differentially enriched genes that regulate myogenic growth and differentiation between the modern and legacy broiler lines. Specifically, differences in the ratios of several positive (IGF1, IGF1R, WFIKKN2) and negative (MSTN, ACE) myogenic growth regulators may help explain the differences underlying the enhanced growth characteristics of the modern broilers.

Effects of Protease, Phytase and a Bacillus sp. Direct-Fed Microbial on Nutrient and Energy Digestibility, Ileal Brush Border Digestive Enzyme Activity and Cecal Short-Chain Fatty Acid Concentration in Broiler Chickens

UNLABELLED A 40-wk experiment was conducted using Hy-Line W-36 laying hens (19-wk old) to investigate the impact of feeding cholecalciferol-enriched diets on egg yolk quality. Feeds were enriched with 4 cholecalciferol levels, 9700 (diet 2), 17200 (diet 3), 24700 (diet 4), and 102200 (diet 5) IU/kg feed. The control (diet 1) contained 2200 IU cholecalciferol/kg feed. Eggs from each replicate group of enriched diets were collected daily and the yolks were pooled into 2-d period during the first 2 wk. During weeks 3, 4, 6, 8, 12, 16, 20, 24, 28, 32, 36, and 40, pooled samples were generated by daily collection of 3 consecutive days of egg production. The cholecalciferol content of egg yolk from the enriched diets increased rapidly during the first 3 wk. The peak cholecalciferol concentrations in egg yolk that occurred at week 3 were 865, 1641, 2411, and 34815 IU/100 g egg yolk (wet basis) from diet 2 to 5. The average cholecalciferol concentration in yolk during weeks 3 to 40 and the deposition rate of cholecalciferol during the first 3 wk were both linearly increased with the dietary cholecalciferol level when the feed contained no more than 24700 IU/kg cholecalciferol. Egg yolk lipid profile (total lipid content, fatty acid composition, phospholipid composition, and unsaponifiables), physical and functional properties (yolk viscosity and emulsifying property), and sensory quality of hard-boiled egg yolk were not affected by the cholecalciferol enrichment in the feed. PRACTICAL APPLICATION A linear dose-response relationship between dietary vitamin D(3) level and egg yolk vitamin D(3) content was established at relatively low enrichment levels. Such relationship can be used to formulate feed to achieve a target egg vitamin D level. High vitamin D yolk showed no difference from the conventional yolk in other compositional, functional, and sensory properties.

Effects of direct-fed microbial supplementation on broiler performance, intestinal nutrient transport and integrity under experimental conditions with increased microbial challenge

Two experiments were conducted to determine the effects of protease and phytase (PP) and a Bacillus sp. direct-fed microbial (DFM) on dietary energy and nutrient utilization in broiler chickens. In the first experiment, Ross 308 broiler chicks were fed diets supplemented with PP and DFM in a 2×2 factorial arrangement. The 4 diets (control (CON), CON + PP, CON + DFM, and CON + PP + DFM) were fed from 15–21 days of age. In Experiment 1, significant interaction (P≤0.01) between PP and DFM on the apparent ileal digestibility coefficient for starch, crude protein, and amino acid indicated that both additives increased the digestibility. Both additives increased the nitrogen retention coefficient with a significant interaction (P≤0.01). Although no interaction was observed, significant main effects (P≤0.01) for nitrogen-corrected apparent ME (AMEn) for PP or DFM indicated an additive response. In a follow-up experiment, Ross 308 broiler chicks were fed the same experimental diets from 1–21 days of age. Activities of ileal brush border maltase, sucrase, and L-alanine aminopeptidase were increased (P≤0.01) by PP addition, while a trend (P = 0.07) for increased sucrase activity was observed in chickens fed DFM, in Experiment 2. The proportion of cecal butyrate was increased (P≤0.01) by DFM addition. Increased nutrient utilization and nitrogen retention appear to involve separate but complementary mechanisms for PP and DFM, however AMEn responses appear to have separate and additive mechanisms.

Effects of increasing concentrations of corn distillers dried grains with solubles on chemical composition and nutrient content of egg

Abstract 1. The effects of Aspergillus oryzae- and Bacillus subtilis-based direct-fed microbials (DFM) were investigated on the performance, ileal nutrient transport and intestinal integrity of broiler chickens, raised under experimental conditions, with increased intestinal microbial challenge. 2. The first study was a 3 × 2 factorial experiment, with 3 dietary treatments (control (CON), CON + DFM and CON + antibiotic growth promoter) with and without challenge. Chicks were fed experimental diets from 1 to 28 d, while the challenge was provided by vaccinating with 10 times the normal dose of commercial coccidial vaccine on d 9. In a second experiment, two groups of 1 d-old broilers, housed on built-up litter (uncleaned from two previous flocks), were fed the same CON and CON + DFM diets from 1 to 21 d. 3. The challenge in the first experiment reduced performance, but no differences were observed among dietary treatments from 8 to 28 d. The challenge reduced the ileal epithelial flux for D-glucose, L-lysine, DL-methionine and phosphorus on d 21. Epithelial flux for D-glucose, L-lysine and DL-methionine were increased by DFM. Ileal trans-epithelial electrical resistance (TER) was increased in challenged broilers fed DFM, although this was not observed in unchallenged birds as indicated by a significant interaction. 4. Ileal mucin mRNA expression and colon TER were increased, and colon endotoxin permeability was reduced by DFM on d 21 in the second experiment. 5. It was concluded that the addition of DFM in the diet improved the intestinal integrity of broiler chickens raised under experimental conditions designed to provide increased intestinal microbial challenge.

Validation of the effects of small differences in dietary metabolizable energy and feed restriction in first-cycle laying hens

The objective of this study was to determine the effects of feeding high concentrations of corn distillers dried grains with solubles (DDGS) on chemical composition and selected nutrient content of egg yolk. Four isocaloric diets were formulated to contain 0, 17, 35, or 50% corn DDGS. A total of two hundred forty 54-wk-old Single-Comb White Leghorn laying hens were randomly allotted to 2 birds per cage with 3 consecutive cages representing an experimental unit (EU). Each EU was randomly assigned to 1 of 4 dietary treatments according to a completely randomized design. Hens were fed for a 24-wk experimental period after transition feeding to gradually increase corn DDGS inclusion over a 4-wk period. Two sets of experimental diets were formulated to meet or exceed the NRC nutrient recommendations for laying hens. Each diet formula was fed for 12 wk. Chemical composition and nutritional components in egg yolk were measured every 2 wk. The results showed that egg yolk from hens fed a DDGS-containing diet tended to have higher fat content and lower protein content. Total polyunsaturated fatty acids were significantly increased by the DDGS diet. The contents of choline and cholesterol were initially higher in the 50% DDGS treatment group, but were not different in the later period, especially during the last 4 wk. Lutein content increased linearly as DDGS level increased. The results indicated that feeding a high level of DDGS can increase the content of lutein and polyunsaturated fatty acids in egg yolk, but may not affect the content of cholesterol and choline.

Effects of increasing concentrations of corn distillers dried grains with solubles on the egg production and internal quality of eggs

An experiment was conducted to evaluate energy utilization of laying hens fed diets containing 2 ME concentrations, using response criteria including performance, BW, abdominal fat pad, and energy digestibility. The experiment was a 2 × 2 factorial with 2 feeding regimens (ad libitum and restriction fed), and 2 dietary ME levels [2,880 kcal/kg of ME (CON); and 2,790 kcal/kg of ME (LME)]. A total of 60 Hy-Line W36 first-cycle laying hens were fed experimental diets, resulting in 15 individually caged hens for each of the 4 treatments. Hens in the restriction-fed group were fed 90 g of feed per day. The CON diet was formulated to meet or exceed the NRC (1994) recommendations with 2,880 kcal/kg, whereas the LME diet was similar with the exception of a 90 kcal/kg reduction in ME. Hens were fed experimental diets for 12 wk from hen 28 to 39 wk of age. Hen day egg production, weekly feed intake, and every 2 wk, egg weights and egg mass were recorded, whereas hen BW was measured every 4 wk. Excreta samples were collected over the last 5 d of experiment to determine AMEn. Abdominal fat pads were measured individually for all hens at the end of experiment. There were no interactions between feeding regimens and dietary ME levels throughout the experiment. Feed restriction resulted in reductions (P ≤ 0.01) in hen day egg production, BW, and abdominal fat pad, indicating reduced nutrient availability to partition toward production, maintenance, and storage functions. The reduction in energy intake between CON and LME fed birds (90 kcal/kg) did not change the energy partitioned toward production or maintenance, but reduced (P = 0.03) the energy stored (reduced fat pad) of LME-fed hens. These results suggest that energy is used following the pattern of production and maintenance before storage requirements and that fat pad (energy storage) may be the most sensitive indicator of dietary energy status over short-term in Hy-Line W36 laying hens.

Unique genetic responses revealed in RNA-seq of the spleen of chickens stimulated with lipopolysaccharide and short-term heat

A study was conducted to determine the effect of feeding high concentrations of corn distillers dried grains with solubles (DDGS) on egg production and the internal quality of eggs from laying hens. Four diets were formulated to contain 0, 17, 35, or 50% corn DDGS. A total of two hundred forty 54-wk-old Single-Comb White Leghorn laying hens were randomly allotted to 2 birds per cage with 3 consecutive cages representing an experimental unit (EU). Each EU was assigned to 1 of 4 dietary treatments according to a completely randomized design. Hens were fed for a 24-wk experimental period after transition feeding to gradually increase corn DDGS inclusion over a 4-wk period. Two sets of experimental diets were formulated, and each diet was fed for 12 wk. Egg production, feed consumption, egg component, yolk color, Haugh unit during storage times, and shell breaking strength were measured. Egg production, egg weight, egg mass, feed intake, and feed efficiency were adversely affected by the highest level of DDGS in the diet (50%) during the first 12-wk period. Once diets were reformulated to include an increased concentration of both lysine and methionine, differences among the dietary treatments were reduced, as the performance of the 50% DDGS diets was greatly improved. Over the last 6 wk of study, no differences in egg production, egg weight, and feed intake among DDGS treatments were found. The DDGS diets positively affected the internal quality of eggs during storage. Improved yolk color and Haugh unit were observed as the dietary DDGS levels increased, but the increase for Haugh unit was significant only when the DDGS level was 50%. Shell weight percentage was increased in the 50% DDGS diet, but no differences in yolk and albumen percentage were observed. It was concluded that up to 50% of DDGS could be included in the layers diet without affecting egg weight, feed intake, egg mass, feed efficiency, and egg production as long as digestible amino acids were sufficient in DDGS-added diets.

Effects of long-term supplementation of laying hens with high concentrations of cholecalciferol on performance and egg quality

Climate change and disease have large negative impacts on poultry production, but little is known about the interactions of responses to these stressors in chickens. Fayoumi (heat and disease resistant) and broiler (heat and disease susceptible) chicken lines were stimulated at 22 days of age, using a 2x2x2 factorial design including: breed (Fayoumi or broiler), inflammatory stimulus (lipopolysaccharide (LPS) or saline), and temperature (35°C or 25°C). Transcriptional changes in spleens were analyzed using RNA-sequencing on the Illumina HiSeq 2500. Thirty-two individual cDNA libraries were sequenced (four per treatment) and an average of 22 million reads were generated per library. Stimulation with LPS induced more differentially expressed genes (DEG, log2 fold change ≥ 2 and FDR ≤ 0.05) in the broiler (N = 283) than the Fayoumi (N = 85), whereas heat treatment resulted in fewer DEG in broiler (N = 22) compared to Fayoumi (N = 107). The double stimulus of LPS+heat induced the largest numbers of changes in gene expression, for which broiler had 567 DEG and Fayoumi had 1471 DEG of which 399 were shared between breeds. Further analysis of DEG revealed pathways impacted by these stressors such as Remodelling of Epithelial Adherens Junctions due to heat stress, Granulocyte Adhesion and Diapedesis due to LPS, and Hepatic Fibrosis/Hepatic Stellate Cell Activation due to LPS+heat. The genes and pathways identified provide deeper understanding of the response to the applied stressors and may serve as biomarkers for genetic selection for heat and disease tolerant chickens.