Muscle fiber cross-sectional area (CSA) and density of the pectoralis major muscle on the different quail lines at 42 and 75 d post-hatch.

a–b<p>Different superscripts in the each muscle region represent significant differences (<i>P</i><0.05).</p><p>Abbreviations: LW, low weight; RBC, random bred control.</p

Expression of the different myosin heavy chain (MHC) isoforms and myogenic factors in the low weight (LW) and random bred control (RBC) quail lines.

<p>Time point comparison of the neonatal MHC isoform using 2E9 monoclonal antibody (A), adult MHC isoform using AB8 monoclonal antibody (B), Pax7 (E), MyoD (F), and Myf-5 (I) between the LW and RBC quail lines (1 quail per each time point). Quail line comparison of neonatal MHC isoforms at 3 and 75 d post-hatch (C), adult MHC isoform at 28, 42, and 75 d post-hatch (D), Pax7 at 7 and 14 d post-hatch (G), MyoD at 0, 3, and 7 d post-hatch (H), and Myf-5 at 3 and 7 d post-hatch (J) (3 quail per each time point and each quail line). Expressions of myogenin mRNA in the two quail lines at 0, 3, 7, 14, 28, 42, and 75 d post-hatch (K). Total RNA was isolated from the pectoralis major muscle. Expressions of myogenin were measured by quantitative real-time PCR with avian ribosomal protein 13 (RPS13) as a control for normalization. Protein staining of the different quail lines at each time point (L; 1 quail per each time point) and at 0, 3, 7, 14, 28, 42, and 75 d post-hatch (M; 3 quail per each time point and each quail line). Gels used as a control of protein loading. Bars indicate standard errors. Level of significance: NS, not significant; * <i>P</i><0.05; ** <i>P</i><0.01; *** <i>P</i><0.001.</p

Blood NEFA levels and the expression of G0S2 and ATGL in fat tissues in different feeding conditions.

<p>(<b>A</b>) and (B) The levels of NEFA in serum were significantly lower in transgenic quail than in non-transgenic quail under the short-term fasting and feed restriction conditions (<i>n</i>≥5). AL: <i>ad libitum</i>, F4H: fasting for 4 hours, and FR2W: feed restriction for 2 weeks. (C) and (D) The expression of G0S2, ATGL, and FABP4 in fat tissues depending on feeding conditions, <i>ad libitum</i> (AL) vs. fasting for 12 hours (F12H). G0S2 was continuously higher in transgenic quail than non-transgenic quail. The expression of ATGL was higher in fasting condition in both transgenic and non-transgenic quail. The ratio of ATGL to G0S2 was highest in non-transgenic quail under the fasting condition. The expression of FABP4 was not significantly variable regardless of feeding condition. Values are represented as mean ± SEM. *, **, and *** indicate significance levels of <i>P</i><0.05, 0.01, and 0.001, respectively. NS means non-significant.</p

Lentiviral vector construct and its expression in adipose tissues of transgenic quail.

<p>(A) Diagram of lentiviral vector used for producing transgenic quail. The vector contains 2 kb promoter of chicken fatty acid binding protein and chicken G0S2 coding DNA sequences. The primers for detecting the transgene are presented as ^f1 for forward primer (LTG0S2-F) and ^r1 and ^r2 for reverse primers (LT-R and WPRE-R, respectively). (B) and (<b>C</b>) The expression level of G0S2 protein in adipose tissues of adult transgenic (Tg) and non-transgenic (Non-Tg) quail. Values are represented as mean ± SEM (n = 3). * indicates significance level of <i>P</i><0.05.</p

Comparison of embryo weight (A) and embryo percentage relative to egg weight (B) between the low weight (LW) and random bred control (RBC) quail lines.

<p>Bars indicate standard errors. Level of significance: * <i>P</i><0.05; *** <i>P</i><0.001.</p

Muscle fiber cross-sections and centered nuclei in the pectoralis major muscle between the low weight (LW) and random bred control (RBC) quail lines at 42 d post-hatch that were stained for hematoxylin and eosin (A); Comparison of centered nuclei percentage relative to total counted nuclei between LW and RBC quail lines at 42 d post-hatch (B).

<p>Arrows indicate centered nuclei. Bars indicate standard errors. Level of significance: *** <i>P</i><0.001.</p

Inhibition of Lipolysis in the Novel Transgenic Quail Model Overexpressing G₀/G₁ Switch Gene 2 in the Adipose Tissue during Feed Restriction

<div><p>In addition to the issue of obesity in humans, the production of low-fat meat from domestic animals is important in the agricultural industry to satisfy consumer demand. Understanding the regulation of lipolysis in adipose tissue could advance our knowledge to potentially solve both issues. Although the G₀/G₁ switch gene 2 (G0S2) was recently identified as an inhibitor of adipose triglyceride lipase (ATGL) <i>in vitro</i>, its role <i>in vivo</i> has not been fully clarified. This study was conducted to investigate the role of G0S2 gene <i>in vivo</i> by using two independent transgenic quail lines during different energy conditions. Unexpectedly, G0S2 overexpression had a negligible effect on plasma NEFA concentration, fat cell size and fat pad weight under <i>ad libitum</i> feeding condition when adipose lipolytic activity is minimal. A two-week feed restriction in non-transgenic quail expectedly caused increased plasma NEFA concentration and dramatically reduced fat cell size and fat pad weight. Contrary, G0S2 overexpression under a feed restriction resulted in a significantly less elevation of plasma NEFA concentration and smaller reductions in fat pad weights and fat cell size compared to non-transgenic quail, demonstrating inhibition of lipolysis and resistance to loss of fat by G0S2. Excessive G0S2 inhibits lipolysis <i>in vivo</i> during active lipolytic conditions, such as food restriction and fasting, suggesting G0S2 as a potential target for treatment of obesity. In addition, transgenic quail are novel models for studying lipid metabolism and mechanisms of obesity.</p></div

Analysis of growth and organ weights of transgenic and non-transgenic quail fed <i>ad libitum</i>.

<p>(A and B) Growth curve of FG1 and FG3 lines of transgenic and non-transgenic quail fed <i>ad libitum</i>. Body weights of male transgenic and non-transgenic quail were measured every week from hatch to 8 weeks of age. (C and D) Organ weights of FG1 quail fed <i>ad libitum</i>. Organ weights were measured and calculated to the percentage of total body weight. There was no difference between transgenic and non-transgenic quail in the tissue and organ weights (<i>n</i> = 5). SF: subcutaneous fat, AF: abdominal fat, LPM: left pectoralis muscle, H; heart, Li: liver, Lu: lung, and K: kidney. (E and F) Organ weights of FG3 quail fed <i>ad libitum</i>. FG3 did not show a difference in organ weight between transgenic (<i>n</i> = 6) and non-transgenic (<i>n</i> = 8) quail under the <i>ad libitum</i> feeding. Values are represented as mean ± SEM.</p

Analysis of body and organ weights of transgenic and non-transgenic quail that underwent feed restriction.

<p>(A) Changes of body weights after feed restriction for two weeks. Non-transgenic quail lost more body weight than transgenic quail in both lines. (B and C) Weights of fat pads after feed restriction. Both transgenic quail of FG1 and FG3 had significantly greater fat pads than non-transgenic quail after feed restriction. FR2W: feed restriction for 2 weeks. (D and E) Organ weights after feed restriction. There was no difference in non-adipose organ weights between transgenic and non-transgenic quail after feed restriction. <i>n</i> = 6 for each. Values are represented as mean ± SEM. *, **, and *** indicate significance levels of <i>P</i><0.05, <i>P</i><0.01, and <i>P</i><0.001, respectively.</p

Muscle fiber cross-sections in the pectoralis major muscle between the low weight (LW) and random bred control (RBC) quail lines at 42 (A) and 75 (B) d post-hatch that were stained for myosin ATPase activity after acidic preincubation (pH 4.6).

<p>Bar indicates 100 µm. Abbreviations: IIA, fiber type IIA (fast-twitch and oxido-glycolytic); IIB, fiber type IIB (fast-twitch and glycolytic).</p