Representative spot maps of M (Muscovy) and PK (Pekin duck).

<p><b>HT</b> stands for heat treated and <b>RC</b> stands for recovery after heat stress. Equal amounts of protein (850 µg) were loaded and separated on 17 cm IPG strips (PH 3-10), followed by electrophoresis on 12.5% SDS-PAGE gels for second dimension electrophoresis. The gels were stained with CCB G250.</p

2-DE patterns of proteins extracted from Muscovy (A) and Pekin ducks (B).

<p>The experiment was repeated 3 times, and 54 differentially expressed proteins showing significant spot intensity changes under heat stress and recovery are marked in A and B. The proteins to which these 54 differentially expressed protein spots correspond are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076917#pone-0076917-t002" target="_blank">Table 2</a>.</p

Comparative Proteomic Analysis of the Hepatic Response to Heat Stress in Muscovy and Pekin Ducks: Insight into Thermal Tolerance Related to Energy Metabolism

<div><p>The Pekin duck, bred from the mallard (<i>Anas platyrhynchos</i>) in china, is one of the most famous meat duck species in the world. However, it is more sensitive to heat stress than Muscovy duck, which is believed to have originated in South America. With temperature raising, mortality, laying performance, and meat quality of the Pekin duck are severely affected. This study aims to uncover the temperature-dependent proteins of two duck species using comparative proteomic approach. Duck was cultured under 39°C ± 0.5°C for 1 h, and then immediately returned to 20°C for a 3 h recovery period, the liver proteins were extracted and electrophoresed in two-dimensional mode. After analysis of gel images, 61 differentially expressed proteins were detected, 54 were clearly identified by MALDI TOF/TOF MS. Of the 54 differentially expressed protein spots identified, 7 were found in both species, whereas 47 were species specific (25 in Muscovy duck and 22 in Pekin duck). As is well known, chaperone proteins, such as heat shock protein (HSP) 70 and HSP10, were abundantly up-regulated in both species in response to heat stress. However, we also found that several proteins, such as α-enolase, and <i>S</i>-adenosylmethionine synthetase, showed different expression patterns in the 2 duck species. The enriched biological processes were grouped into 3 main categories according to gene ontology analysis: cell death and apoptosis (20.93%), amino acid metabolism (13.95%) and oxidation reduction (20.93%). The mRNA levels of several differentially expressed protein were investigated by real-time RT-PCR. To our knowledge, this study is the first to provide insights into the differential expression of proteins following heat stress in ducks and enables better understanding of possible heat stress response mechanisms in animals.</p> </div

Possible energy metabolism in duck.

<p>The symbols “↑” and “↓” indicate the protein up- or down-regulation response to heat stress. Species-specific changes are indicated as M (Muscovy) or PK (Pekin duck). <b>Abbreviations: UDPG</b>, UDP-glucose; <b>Gn</b>, glycogen; <b>G3P</b>, glyceraldehyde-3-phosphate; <b>3-PG</b>, 3-phosphoglycerate; <b>PEP</b>, phosphoenolpyruvate; <b>CoA</b>, coenzyme A; <b>ETC</b>, electron transport chain.</p

Comparison of expression patterns between Muscovy (M) and Pekin ducks (PK) for 5 important heat-responsive differentially expressed protein spots under heat stress and recovery.

<p>A: Magnified views of 5 important proteins marked in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076917#pone-0076917-g002" target="_blank">Figure 2</a>. B: Expression patterns of the 5 proteins. Spot 9: HSP70; Spot 16: <i>S</i>-adenosylmethionine synthase isoform type-1; Spot 17: Alpha-enolase; Spot 24: catalase; Spot 39: superoxide dismutase (Cu-Zn).</p

Validation of the differential expression of 8 proteins at mRNA levels by RT-PCR.

<p>The experimental procedure and the statistical estimation for the parallel runs are described in “Material and methods”. Data was expressed as mean ± SEM (6 Pekin ducks and 6 Muscovy ducks for all groups). Statistical significance was determined using the Student’s t test with two-tailed <i>P</i> values. * indicates <i>P</i> < 0.05, ** indicates <i>P</i> < 0.01. <b>Abbreviations: SAMS1</b>, <i>S</i>-adenosylmethionine synthase isoform type-1; <b>ENO1</b>, alpha-enolase; <b>CAT</b>, catalase; <b>PHB</b>, prohibitin; <b>SOD1</b>, superoxide dismutase (Cu-Zn).</p

Classification of identified proteins by gene ontology (GO) enrichment analysis and tissue mapping.

<p>A: biological processes. B: tissue mapping. C: molecular function. D: cellular component.</p

CD-1 mice body weights following various treatment groups.

<p>The groups include: control, al libitum feeding with exercise (AL+Exe), pair feeding with exercise (PF+Exe) and dietary calorie restriction (DCR). Animals were adapted to surroundings and underwent treadmill training during the first two weeks. The arrow indicates the beginning of dietary calorie restriction and physical exercise. Results presented as mean ± SE, n = 10–13 animals/group. *p<0.05 (compared to control), **p<0.01 (compared to control). For side asterisks: * (compared to control group), *’ (compared to PF+Exe).</p

Plasma DG (normalized MS intensity per μl) following various treatments.

<p>A) Total plasma DG from all treatment groups. B) Individual plasma DG acyl group for all treatments. Presented species contain at least one of the listed fatty acids. The fatty acids presented in the figure were the most abundant within samples. Normalization is to the intensity of the DG internal standard. Data presented as mean± SE, n = 10–13 animals/group. *p<0.05 as compared to control.</p

TG and DG molecular species following various treatments.

<p>A) Plasma TG. B) Tissue TG. C) Plasma DG. D) Tissue DG. For TG (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116398#pone.0116398.g002" target="_blank">Fig. 2A and 2B</a>), one fatty acid is indicated in each panel, and the other two are combined and shown in the horizontal axis. Data presented are mean± SE. n = 10–13 animals/group.</p