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16 pages, 2098 KiB  
Article
Mitochondrial Abundance and Function Differ Across Muscle Within Species
by Con-Ning Yen, Jocelyn S. Bodmer, Jordan C. Wicks, Morgan D. Zumbaugh, Michael E. Persia, Tim H. Shi and David E. Gerrard
Metabolites 2024, 14(10), 553; https://doi.org/10.3390/metabo14100553 - 16 Oct 2024
Abstract
Background: Mitochondria are considered the powerhouse of cells, and skeletal muscle cells are no exception. However, information regarding muscle mitochondria from different species is limited. Methods: Different muscles from cattle, pigs and chickens were analyzed for mitochondrial DNA (mtDNA), protein and [...] Read more.
Background: Mitochondria are considered the powerhouse of cells, and skeletal muscle cells are no exception. However, information regarding muscle mitochondria from different species is limited. Methods: Different muscles from cattle, pigs and chickens were analyzed for mitochondrial DNA (mtDNA), protein and oxygen consumption. Results: Bovine oxidative muscle mitochondria contain greater mtDNA (p < 0.05), protein (succinate dehydrogenase, SDHA, p < 0.01; citrate synthase, CS, p < 0.01; complex I, CI, p < 0.05), and oxygen consumption (p < 0.01) than their glycolytic counterpart. Likewise, porcine oxidative muscle contains greater mtDNA (p < 0.01), mitochondrial proteins (SDHA, p < 0.05; CS, p < 0.001; CI, p < 0.01) and oxidative phosphorylation capacity (OXPHOS, p < 0.05) in comparison to glycolytic muscle. However, avian oxidative skeletal muscle showed no differences in absolute mtDNA, SDHA, CI, complex II, lactate dehydrogenase, or glyceraldehyde 3 phosphate dehydrogenase compared to their glycolytic counterpart. Even so, avian mitochondria isolated from oxidative muscles had greater OXPHOS capacity (p < 0.05) than glycolytic muscle. Conclusions: These data show avian mitochondria function is independent of absolute mtDNA content and protein abundance, and argue that multiple levels of inquiry are warranted to determine the wholistic role of mitochondria in skeletal muscle. Full article
(This article belongs to the Special Issue Unlocking the Mysteries of Muscle Metabolism in the Animal Sciences)
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Figure 1

Figure 1
<p>(<b>A</b>,<b>D</b>,<b>G</b>) Absolute mitochondrial DNA (mtDNA) number in glycolytic and oxidative muscles. (<b>B</b>,<b>E</b>,<b>H</b>) Relative mtDNA compared to genomic DNA (2 <sup>−∆CT</sup>) in glycolytic and oxidative muscles. (<b>C</b>,<b>F</b>,<b>I</b>) Fold change (2 <sup>−∆∆CT</sup>) of mtDNA in oxidative compared to the glycolytic muscle type. (<b>A</b>–<b>C</b>) Bovine (<span class="html-italic">n</span> = 6) and (<b>D</b>–<b>F</b>) porcine (<span class="html-italic">n</span> = 6) muscle mtDNA content from <span class="html-italic">longissimus lumborum</span> (LL) and <span class="html-italic">masseter</span> (MS). (<b>G</b>–<b>I</b>) Avian muscle (<span class="html-italic">n</span> = 6) mtDNA content in <span class="html-italic">pectoralis major</span> (PM) and <span class="html-italic">quadriceps femoris</span> (QF). All values are displayed as least square means followed by standard error bars. Significance is denoted as * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001.</p>
Full article ">Figure 2
<p>Oxidative protein abundance from whole muscle in bovine (<b>A</b>–<b>D</b>), porcine (<b>E</b>–<b>H</b>), and avian (<b>I</b>–<b>L</b>). Bovine (<span class="html-italic">n</span> = 6) and porcine (<span class="html-italic">n</span> = 6) muscle protein content from <span class="html-italic">longissimus lumborum</span> (LL) and <span class="html-italic">masseter</span> (MS). Avian (<span class="html-italic">n</span> = 6) muscle protein content in <span class="html-italic">pectoralis major</span> (PM) and <span class="html-italic">quadriceps femoris</span> (QF). Oxidative protein abundance of (<b>A</b>,<b>E</b>,<b>I</b>) succinate dehydrogenase (SDHA), (<b>B</b>,<b>F</b>,<b>J</b>) citrate synthase (CS), and (<b>C</b>,<b>G</b>,<b>K</b>) voltage-dependent anion channel (VDAC). (<b>D</b>,<b>H</b>,<b>L</b>) Representative Western blot images of SDHA, CS, VDAC, and total protein stain (Ponceau S). All values are displayed as least square means followed by standard error bars. Significance is denoted as * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001.</p>
Full article ">Figure 3
<p>Glycolytic protein abundance from whole muscle in bovine (<b>A</b>–<b>C</b>), porcine (<b>D</b>–<b>F</b>), and avian (<b>G</b>–<b>I</b>). Bovine (<span class="html-italic">n</span> = 6) and porcine (<span class="html-italic">n</span> = 6) muscle protein content from <span class="html-italic">longissimus lumborum</span> (LL) and <span class="html-italic">masseter</span> (MS). Avian muscle (<span class="html-italic">n</span> = 6) protein content in <span class="html-italic">pectoralis major</span> (PM) and <span class="html-italic">quadriceps femoris</span> (QF). Glycolytic enzyme protein abundance of (<b>A</b>,<b>D</b>,<b>G</b>) glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and (<b>B</b>,<b>E</b>,<b>H</b>) lactate dehydrogenase (LDHA). (<b>C</b>,<b>F</b>,<b>I</b>) Representative Western blot images of GAPDH, LDHA, and total protein stain (Ponceau S). All values are displayed as least square means followed by standard error bars. Significance is denoted as ** <span class="html-italic">p</span> &lt; 0.01 and *** <span class="html-italic">p</span> &lt; 0.001.</p>
Full article ">Figure 4
<p>Mitochondrial protein abundance in glycolytic and oxidative muscles from bovine (<b>A</b>–<b>C</b>), porcine (<b>D</b>–<b>F</b>), and avian (<b>G</b>–<b>I</b>) mitochondria enriched fractions. Bovine (<span class="html-italic">n</span> = 6) and porcine (<span class="html-italic">n</span> = 6) mitochondrial protein content from <span class="html-italic">longissimus lumborum</span> (LL) and <span class="html-italic">masseter</span> (MS) muscles. Avian (<span class="html-italic">n</span> = 6) mitochondrial protein content from <span class="html-italic">pectoralis major</span> (PM) and <span class="html-italic">quadriceps femoris</span> (QF) muscles. (<b>A</b>,<b>D</b>,<b>G</b>) Mitochondrial proteins abundance of complex I (CI, NDUFB8) and (<b>B</b>,<b>E</b>,<b>H</b>) complex II (CII, SDHB) and (<b>C</b>,<b>F</b>,<b>I</b>) voltage dependent anion channel (VDAC). (<b>J</b>) Representative Western blot images of complex I, complex II, complex III, complex V, VDAC, and total protein stain (Ponceau S). All values are displayed as least square means followed by standard error bars. Significance is denoted as † <span class="html-italic">p =</span> 0.08, * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01.</p>
Full article ">Figure 5
<p>Oxygen consumption rate of mitochondria isolated from (<b>A</b>,<b>D</b>) bovine (<span class="html-italic">n</span> = 6) and (<b>B</b>,<b>E</b>) porcine (<span class="html-italic">n</span> = 6) <span class="html-italic">longissimus lumborum</span> (LL) and <span class="html-italic">masseter</span> (MS) and (<b>C</b>,<b>F</b>) avian (<span class="html-italic">n</span> = 8) <span class="html-italic">pectoralis major</span> (PM) and <span class="html-italic">quadriceps femoris</span> (QF) muscles under saturating concentrations of pyruvate/malate (PyM; <b>A</b>–<b>C</b>) and succinate/rotenone (SR; <b>D</b>–<b>F</b>) substrates. Baseline represents basal respiration of isolated mitochondria with substrates. OXPHOS capacity is ADP (5 mM) stimulated respiration. Proton leak is determined with 2 µM oligomycin. Maximal respiration is achieved with the uncoupler FCCP (4 µM). All values are displayed as least square means followed by standard error bars. Significance is denoted as * <span class="html-italic">p</span> &lt; 0.05 and ** <span class="html-italic">p</span> &lt; 0.01.</p>
Full article ">Figure 6
<p>Oxygen consumption rate of mitochondria isolated from (<b>A</b>,<b>D</b>) bovine (<span class="html-italic">n</span> = 6) and (<b>B</b>,<b>E</b>) porcine (<span class="html-italic">n</span> = 6) <span class="html-italic">longissimus lumborum</span> (LL) and <span class="html-italic">masseter</span> (MS) and (<b>C</b>,<b>F</b>) avian <span class="html-italic">pectoralis major</span> (PM, <span class="html-italic">n</span> = 10) and <span class="html-italic">quadriceps femoris</span> (QF, <span class="html-italic">n</span> = 9) muscles under saturating concentrations of glutamate/malate (GM; <b>A</b>–<b>C</b>) and palmitoyl-carnitine/malate (PCM; <b>D</b>–<b>F</b>) substrates. Baseline represents basal respiration of isolated mitochondria with substrates. OXPHOS capacity is ADP (5 mM) stimulated respiration. Proton leak is determined with 2 µM oligomycin. Maximal respiration is achieved with the uncoupler FCCP (4 µM). All values are displayed as least square means followed by standard error bars. Significance is denoted as * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, and *** <span class="html-italic">p</span> &lt; 0.001.</p>
Full article ">
21 pages, 3484 KiB  
Article
In Vivo Study of the Effect of Sugarcane Bagasse Lignin Supplementation on Broiler Chicken Diet as a Step to Validate the Established Chicken Gastrointestinal Tract In Vitro Model
by Nelson Mota de Carvalho, Carla Giselly de Souza, Célia Maria Costa, Cláudia Castro, Joana F. Fangueiro, Bruno Horta, Divanildo Outor-Monteiro, José Teixeira, José Luís Mourão, Victor Pinheiro, Ana L. Amaro, Patrícia Santos Costa, Catarina S. S. Oliveira, Manuela Estevez Pintado, Diana Luazi Oliveira and Ana Raquel Madureira
Sustainability 2024, 16(20), 8946; https://doi.org/10.3390/su16208946 - 16 Oct 2024
Abstract
Since the global restrictions on antibiotics in poultry systems, there has been a growing demand for natural and sustainable feed additives for disease prevention and poultry nutrition. This study evaluated the effects of incorporating sugarcane bagasse (SCB) lignin into broiler chicken diets. The [...] Read more.
Since the global restrictions on antibiotics in poultry systems, there has been a growing demand for natural and sustainable feed additives for disease prevention and poultry nutrition. This study evaluated the effects of incorporating sugarcane bagasse (SCB) lignin into broiler chicken diets. The performance of the chickens, including body weight, feed intake, and mortality, as well as intestinal histomorphometry, and cecum content pH, microbiota, and volatile fatty acids were assessed. In addition, we also aimed to validate an in vitro gastrointestinal tract (GIT) model developed by Carvalho et al. (2023). One hundred and eight 1-day-old Ross 308 chicks were randomly and equally divided into two groups. The first group was fed a basal diet (BD group), while the second group was fed a basal diet supplemented with 1% (w/w) SCB lignin (BD + SCB lignin group) for 36 days. The in vivo conditions of the chicken GIT were replicated in an in vitro model. In the in vivo study, SCB lignin increased cecum acetate and butyrate levels while reducing Bifidobacterium and Enterobacteriaceae, without affecting productivity (body weight, feed intake, and mortality). The in vitro assessment reflected microbiota trends observed in vivo, although without statistical significance. The divergence in organic acid production between the in vivo and in vitro conditions likely resulted from issues with inoculum preparation. This study demonstrates that SCB lignin incorporation positively influences cecal microbiota composition without impacting the animals’ productivity and physiology, suggesting its potential as a functional feed additive. For a more reliable in vitro model, adjustments in inoculum preparation are necessary. Full article
(This article belongs to the Special Issue By-Products of the Agri-Food Industry: Use for Food Fortification)
Show Figures

Figure 1

Figure 1
<p>Animal <span class="html-italic">in vivo</span> assay work flowchart for testing the basal diet (BD) without and with sugarcane bagasse (SCB) lignin supplementation.</p>
Full article ">Figure 2
<p>Image of ileum sample and measurements.</p>
Full article ">Figure 3
<p>(<b>a</b>) Bacterial viable cell counts (log (CFU/mL), mean ± SD) of the cecal contents in different culture media and (<b>b</b>) pH values of the cecal contents diluted at 10% (<span class="html-italic">w</span>/<span class="html-italic">v</span>) in phosphate-buffered saline (PBS) obtained from the chickens fed with a basal diet (BD), without and with sugarcane bagasse (SCB) lignin supplementation. <sup>a,b</sup> Means in the culture media and pH value between sampling times, marked with the same letter, do not differ from each other (<span class="html-italic">p</span> &gt; 0.05). <sup>A,B</sup> Means in culture media and pH values between sampling times for each treatment, marked with the same letter, do not differ from each other (<span class="html-italic">p</span> &gt; 0.05). CBA—Columbia blood agar; MCA—MacConkey agar; MRSA—de Man, Rogosa, and Sharpe agar.</p>
Full article ">Figure 4
<p>Bacterial quantification (log (CFU/mL), means ± SD) of the different bacterial populations in cecal contents obtained from the broiler chickens fed with a basal diet (BD), with or without sugarcane bagasse (SCB) lignin supplementation: (<b>a</b>) Firmicutes, (<b>b</b>) Bacteroidetes, (<b>c</b>) <span class="html-italic">Lactobacillus</span> group, (<b>d</b>) <span class="html-italic">Bacteroides</span>, (<b>e</b>) <span class="html-italic">Bifidobacterium</span>, and (<b>f</b>) <span class="html-italic">Enterobacteriaceae</span> family. <sup>a,b</sup> Means within the bacterial group across sampling times, marked with the same letter, do not differ from each other (<span class="html-italic">p</span> &gt; 0.05). <sup>A,B</sup> Means within the bacterial group across sampling times for each treatment, marked with the same letter, do not differ from the other (<span class="html-italic">p</span> &gt; 0.05).</p>
Full article ">Figure 5
<p>Concentration (mM, means ± SD) of the SCFAs and BCFAs present in the cecum contents obtained from the broiler chickens fed with a basal diet (BD), with or without sugarcane bagasse (SCB) lignin: (<b>a</b>) acetate, (<b>b</b>) propionate, (<b>c</b>) butyrate, (<b>d</b>) isobutyrate, (<b>e</b>) valerate, and (<b>f</b>) isovalerate. <sup>a,b</sup> Means within the organic acid across each sampling times marked with the same letter do not differ from each other (<span class="html-italic">p</span> &gt; 0.05). <sup>A,B</sup> Means within the organic acid across sampling times for each treatment, marked with the same letter, do not differ from each other (<span class="html-italic">p</span> &gt; 0.05).</p>
Full article ">Figure 6
<p>Bacterial quantification (log (CFU/mL), means ± SD) of the different bacterial populations in cecal fermentations for the condition inoculum control (IC), basal diet (BD), with and without sugarcane bagasse (SCB) lignin supplementation: (<b>a</b>) Firmicutes, (<b>b</b>) Bacteroidetes, (<b>c</b>) <span class="html-italic">Lactobacillus</span> group, (<b>d</b>) <span class="html-italic">Bacteroides</span>, (<b>e</b>) <span class="html-italic">Bifidobacterium</span>, and (<b>f</b>) <span class="html-italic">Enterobacteriaceae</span> family. <sup>a,b,c</sup> Means of each bacterial concentration at a given sampling time, marked with the same letter, do not differ from each other (<span class="html-italic">p</span> &gt; 0.05).</p>
Full article ">Figure 7
<p>Concentrations (mM, mean ± SD) of the different organic acids produced during the cecal fermentation for the condition inoculum control (IC), basal diet (BD), with and without sugarcane bagasse (SCB) lignin supplementation: (<b>a</b>) lactate, (<b>b</b>) acetate, (<b>c</b>) propionate, and (<b>d</b>) butyrate. <sup>a,b</sup> Means of each organic acid at a given sampling time, marked with the same letter, do not differ from each other (<span class="html-italic">p</span> &gt; 0.05).</p>
Full article ">Figure 8
<p>Concentration of total ammonia nitrogen (mM, means ± SD) produced during cecal fermentation for the condition inoculum control (IC), basal diet (BD), with and without sugarcane bagasse (SCB) lignin supplementation. <sup>a,b</sup> Means of total ammonia nitrogen at a given sampling time, marked with the same letter, do not differ from each other (<span class="html-italic">p</span> &gt; 0.05).</p>
Full article ">
18 pages, 5966 KiB  
Article
Co-Regulation Mechanism of Host p53 and Fos in Transcriptional Activation of ILTV Immediate-Early Gene ICP4
by Zheyi Liu, Xuefeng Li, Lu Cui, Shufeng Feng, Zongxi Han, Yu Zhang, Shengwang Liu and Hai Li
Microorganisms 2024, 12(10), 2069; https://doi.org/10.3390/microorganisms12102069 (registering DOI) - 16 Oct 2024
Viewed by 48
Abstract
Infectious laryngotracheitis virus (ILTV) exhibits a cascade expression pattern of encoded genes, and ICP4 is the only immediate-early gene of ILTV, which plays a crucial role in initiating the subsequent viral genes. Therefore, studying the transcriptional regulation mechanism of ICP4 holds promise for [...] Read more.
Infectious laryngotracheitis virus (ILTV) exhibits a cascade expression pattern of encoded genes, and ICP4 is the only immediate-early gene of ILTV, which plays a crucial role in initiating the subsequent viral genes. Therefore, studying the transcriptional regulation mechanism of ICP4 holds promise for effectively blocking ILTV infection and spread. Host transcriptional factors p53 and Fos are proven to regulate a variety of viral infections, and our previous studies have demonstrated their synergistic effects in regulating ILTV infection. In this study, we constructed eukaryotic expression vectors for p53 and Fos as well as their specific siRNAs and transfected them into a chicken hepatoma cell line. The results showed that knocking down p53 or Fos significantly inhibited ICP4 transcription, while overexpressing p53 or Fos had an opposite effect. A further CoIP and ChIP-qPCR assay suggested p53 and Fos physically interacted with each other, and jointly bound to the upstream transcriptional regulatory region of ICP4. To elucidate the specific mechanisms of p53 and Fos in regulating ICP4 transcription, we designed p53 and Fos protein mutants by mutating their DNA binding domains, which significantly reduced their binding ability to DNA without affecting their interaction. The results showed that Fos directly bound to the promoter region of ICP4 as a binding target of p53, and the p53–Fos protein complex acted as a transcriptional co-regulator of ICP4. Studying the transcriptional process and regulatory pattern of ICP4 is of great significance for understanding the molecular mechanism of ILTV infection, and thus for finding effective methods to control and prevent it. Full article
(This article belongs to the Special Issue State-of-the-Art Veterinary Microbiology in China (2023, 2024))
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Figure 1

Figure 1
<p>Effects of p53 and Fos overexpression and knockdown on the transcription of <span class="html-italic">ICP4.</span> (<b>A</b>–<b>E</b>) 24 h after instantaneous transfection of pCAG-p53-Flag and pCAG-Fos-HA into LMH cells, the overexpression efficiency of p53 and Fos was analyzed on mRNA level by RT-qPCR (<b>A</b>,<b>B</b>) and on protein level by Western blot (<b>C</b>,<b>D</b>) and immunofluorescence (<b>E</b>). The scale bar indicates 150 µm. (<b>F</b>,<b>G</b>) The knockdown efficiency of p53 and Fos was analyzed on mRNA level by RT-qPCR 24 h after sip53 and siFos transfection into LMH cells. (<b>H</b>,<b>I</b>) 24 h after transfection with pCAG-Flag, pCAG-p53-Flag or pCAG-HA, pCAG-Fos-HA, LMH cells were infected with ILTV (MOI = 1). The transcription level of ILTV immediate-early gene <span class="html-italic">ICP4</span> was detected at the indicated time points by absolute quantitative PCR with standard curve method. (<b>J</b>,<b>K</b>) 24 h after transfection with sicontrol, sip53 or siFos, LMH cells were infected with ILTV (MOI = 1). The transcription level of ILTV immediate-early gene <span class="html-italic">ICP4</span> was quantitatively detected at the indicated time points. Data in (<b>A</b>,<b>B</b>,<b>F</b>–<b>K</b>) are presented as the mean ± SD, <span class="html-italic">n</span> = 3. * <span class="html-italic">p</span> &lt; 0.05 and ** <span class="html-italic">p</span> &lt; 0.01 indicate the levels of significance.</p>
Full article ">Figure 2
<p>p53 promotes <span class="html-italic">Fos</span> transcription and directly binds to the promoter region of <span class="html-italic">Fos</span>. (<b>A</b>,<b>B</b>) Effect of overexpression or knockdown of p53 on <span class="html-italic">Fos</span> transcription was assayed by RT-qPCR. (<b>C</b>,<b>D</b>) Effect of overexpression or knockdown of Fos on <span class="html-italic">p53</span> transcription was assayed by RT-qPCR. (<b>E</b>) Prediction of the putative p53 DNA binding sites within the 2000 bp upstream region of <span class="html-italic">Fos</span> gene using our previous ChIP-sequencing data. (<b>F</b>) The binding level of p53 on the predicted sites was validated by ChIP-qPCR. (<b>G</b>) The transcriptional activity of p53 was assayed by dual-luciferase reporter assay. Data in (<b>A</b>–<b>D</b>,<b>F</b>,<b>G</b>) are represented as mean ± standard deviation, <span class="html-italic">n</span> = 3. * <span class="html-italic">p</span> &lt; 0.05 and ** <span class="html-italic">p</span> &lt; 0.01 indicate the levels of significance.</p>
Full article ">Figure 3
<p>p53 indirectly binds to <span class="html-italic">ICP4</span> promoter as a transcriptional regulator. (<b>A</b>) Prediction of the putative Fos DNA binding sites within the 2000 bp upstream region of <span class="html-italic">ICP4</span> gene using Jasper database. (<b>B</b>) The binding level of Fos on the predicted sites was validated by ChIP-qPCR. (<b>C</b>) The transcriptional activity of Fos was assayed by dual-luciferase reporter assay. (<b>D</b>) Co-IP of p53 and Fos in LMH cells with or without ILTV infection (MOI = 1) using antibodies specifically recognizing HA or Flag. IP: Immunoprecipitation; IB: Immunoblotting. (<b>E</b>) The binding level of p53 on the predicted sites was validated by ChIP-qPCR. Data in (<b>B</b>,<b>C</b>,<b>E</b>) are represented as mean ± standard deviation, <span class="html-italic">n</span> = 3. ** <span class="html-italic">p</span> &lt; 0.01 indicates the levels of significance.</p>
Full article ">Figure 4
<p>p53 directly binding with DNA is not necessary for transcriptional regulation of <span class="html-italic">ICP4.</span> (<b>A</b>) Mutation of chicken p53 at the conserved region of DNA binding domain. (<b>B</b>) 24 h after instantaneous transfection of pCAG-p53-Flag and pCAG-pm1-Flag into LMH cells, the overexpression efficiency was analyzed by Western blot. Tubulin was used as the inner control. (<b>C</b>) The binding of wtp53 and pm1 to the classical p53 target genes was detected by ChIP-qPCR. (<b>D</b>) The effects of wtp53 and pm1 on the transcription of classical p53 target genes were assayed by RT-qPCR. (<b>E</b>) Co-IP of p53 and Fos in LMH cells with or without p53 mutation using antibodies specifically recognizing HA or Flag. IP: Immunoprecipitation; IB: Immunoblotting. (<b>F</b>) The binding of wtp53 and pm1 to <span class="html-italic">ICP4</span> promoter was detected by ChIP-qPCR. (<b>G</b>) The binding of Fos to <span class="html-italic">ICP4</span> promoter upon co-overexpression of wtp53 or pm1 was detected by ChIP-qPCR. (<b>H</b>) The transcriptional activities of wtp53 and pm1 were assayed by dual-luciferase reporter assay. (<b>I</b>) LMH cells were transfected with pCAG-Flag, pCAG-p53-Flag, or pCAG-pm1-Flag, and 24 h later infected with ILTV (MOI = 1). The transcription level of <span class="html-italic">ICP4</span> was quantitatively detected by absolute quantitative PCR with standard curve method. Data in (<b>C</b>,<b>D</b>,<b>F</b>–<b>I</b>) are represented as mean ± standard deviation, <span class="html-italic">n</span> = 3. ** <span class="html-italic">p</span> &lt; 0.01 indicate the levels of significance.</p>
Full article ">Figure 5
<p>Fos directly binding with DNA is required for the transcriptional regulation of <span class="html-italic">ICP4</span> by p53. (<b>A</b>) Mutation of chicken Fos at the conserved region of DNA binding domain. (<b>B</b>) 24 h after instantaneous transfection of pCAG-Fos-HA and pCAG-Fm1-HA into LMH cells, the overexpression efficiency was analyzed by Western blot. Tubulin was used as the inner control. (<b>C</b>) The binding of wide-type Fos and Fm1 to the classical Fos target genes was detected by ChIP-qPCR. (<b>D</b>) The effect of wide-type Fos and Fm1 on the transcription of classical Fos target genes was assayed by RT-qPCR. (<b>E</b>) Co-IP of p53 and Fos in LMH cells with or without Fos mutation using antibodies specifically recognizing HA or Flag. IP: Immunoprecipitation; IB: Immunoblotting. (<b>F</b>) The binding of Fos and Fm1 to <span class="html-italic">ICP4</span> promoter was detected by ChIP-qPCR. (<b>G</b>) The binding of p53 to <span class="html-italic">ICP4</span> promoter upon co-overexpression of wide-type Fos and Fm1 was detected by ChIP-qPCR. (<b>H</b>) The transcriptional activities of wide-type Fos and Fm1 were assayed by dual-luciferase reporter assay. (<b>I</b>) LMH cells were transfected with pCAG-HA, pCAG-Fos-HA, or pCAG-Fm1-HA, and 24 h later infected with ILTV (MOI = 1). The transcription level of <span class="html-italic">ICP4</span> was quantitatively detected by absolute quantitative PCR with standard curve method. Data in (<b>C</b>,<b>D</b>,<b>F</b>–<b>I</b>) are represented as mean ± standard deviation, <span class="html-italic">n</span> = 3. * <span class="html-italic">p</span> &lt; 0.05 and ** <span class="html-italic">p</span> &lt; 0.01 indicate the levels of significance.</p>
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<p>Diagram of host p53 and Fos in transcriptional activation of ILTV immediate-early gene <span class="html-italic">ICP4</span>.</p>
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<p>Prediction of the interaction between p53 monomer and Fos monomer.</p>
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<p>Prediction of interaction between p53 tetramer and Fos monomer.</p>
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20 pages, 8288 KiB  
Article
Temporal Changes in Jejunal and Ileal Microbiota of Broiler Chickens with Clinical Coccidiosis (Eimeria maxima)
by Katarzyna B. Miska, Philip M. Campos, Sara E. Cloft, Mark C. Jenkins and Monika Proszkowiec-Weglarz
Animals 2024, 14(20), 2976; https://doi.org/10.3390/ani14202976 (registering DOI) - 15 Oct 2024
Viewed by 241
Abstract
Coccidiosis in broiler chickens continues to be a major disease of the gastrointestinal tract, causing economic losses to the poultry industry worldwide. The goal of this study was to generate a symptomatic Eimeria maxima (1000 oocysts) infection to determine its effect on the [...] Read more.
Coccidiosis in broiler chickens continues to be a major disease of the gastrointestinal tract, causing economic losses to the poultry industry worldwide. The goal of this study was to generate a symptomatic Eimeria maxima (1000 oocysts) infection to determine its effect on the luminal and mucosal microbiota populations (L and M) in the jejunum and ileum (J and IL). Samples were taken from day 0 to 14 post-infection, and sequencing of 16S rRNA was performed using Illumina technology. Infected birds had significantly (p < 0.0001) lower body weight gain (BWG), higher feed conversion ratio (FCR) (p = 0.0015), increased crypt depth, and decreased villus height (p < 0.05). The significant differences in alpha and beta diversity were observed primarily at height of infection (D7). Analysis of taxonomy indicated that J-L and M were dominated by Lactobacillus, and in IL-M, changeover from Candidatus Arthromitus to Lactobacillus as the major taxon was observed, which occurred quicky in infected animals. LEfSe analysis found that in the J-M of infected chickens, Lactobacillus was significantly more abundant in infected (IF) chickens. These findings show that E. maxima infection affects the microbiota of the small intestine in a time-dependent manner, with different effects on the luminal and mucosal populations. Full article
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<p>Concentration of plasma carotenoids in control (C) and <span class="html-italic">Eimeria maxima</span> (IF)-infected chickens (day PI, day post-infection; mean ± SE).</p>
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<p>Crypt elongation following infection with <span class="html-italic">Eimeria maxima</span>. Expression of <span class="html-italic">Olfm4</span> mRNA by in situ hybridization in the jejunum of broiler chickens that were infected at 21 d of age with either 1000 <span class="html-italic">E. maxima</span> oocysts (IF) or sham infected with sterile water (C) and sampled at 0, 3, 5, 7, 10, and 14 d post-infection (PI). All tissues were counterstained with 50% hematoxylin. Images were captured at 40× magnification (n = 4). (<b>A</b>) Crypt depth and (<b>B</b>) villus height was measured on jejunal sections stained for <span class="html-italic">Olfm4</span> by in situ hybridization. Measures were analyzed by infection status using the nonparametric Welch’s one-way test. Significances (<span class="html-italic">p</span> &lt; 0.05) are indicated by asterisks (*).</p>
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<p>Effect of <span class="html-italic">Eimeria maxima</span> infection at days 0, 3, 5, 7, 10, and 14 post-infection (PI) on the alpha diversity indices (<b>A</b>) Shannon’s entropy and (<b>B</b>) Faith’s PD of the mucosal bacterial populations of the jejunum (J-M). Non-infected birds = C, infected birds = IF. Significant (<span class="html-italic">p</span> &lt; 0.05) differences are indicated by asterisks (*).</p>
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<p>Effect of <span class="html-italic">Eimeria maxima</span> infection at days 0, 3, 5, 7, 10, and 14 post-infection (PI) on the alpha diversity indices (<b>A</b>) Shannon entropy, (<b>B</b>) observed features, Faith’s PD (<b>C</b>), and Evenness (<b>D</b>) of the luminal bacterial populations of the ileum (IL-C). Non-infected birds = C, infected birds = IF. Significant (<span class="html-italic">p</span> &lt; 0.05) differences are indicated by asterisks (*).</p>
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<p>Effect of <span class="html-italic">Eimeria maxima</span> at days 0, 3, 5, 7, 10, and 14 post-infection (PI) on the beta diversity of jejunal luminal (<b>A</b>) (J-C) and jejunal mucosa (<b>B</b>) (J-M) bacterial populations using the principal coordinate analysis (PcoA) based on the weighted (<b>A</b>) and unweighted UniFrac (<b>B</b>) distances between groups. Non-infected birds = C, infected birds = IF. Significant (<span class="html-italic">p</span>  &lt;  0.05) differences are indicated by asterisks (*).</p>
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<p>Effect of <span class="html-italic">Eimeria maxima</span> at days 0, 3, 5, 7, 10, and 14 post-infection (PI) on the beta diversity of ileal luminal (<b>A</b>,<b>B</b>) (IL-C) and ileal mucosa (<b>C</b>) (IL-M) bacterial populations using the principal coordinate analysis (PcoA) based on the weighted (<b>A</b>,<b>C</b>) and unweighted UniFrac (<b>B</b>) distances between groups. Non-infected birds = C, infected birds = IF. Significant (<span class="html-italic">p</span> &lt; 0.05) differences are indicated by asterisks (*).</p>
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<p>Effect of <span class="html-italic">Eimeria maxima</span> at days 0, 3, 5, 7, 10, and 14 post-infection (PI) on relative bacterial abundance (%) at the genus level in the (<b>A</b>) jejunal lumen (J-C) and (<b>B</b>) jejunal mucosa (J-M). Non-infected birds = C, infected birds = IF.</p>
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<p>Effect of <span class="html-italic">Eimeria maxima</span> at days 0, 3, 5, 7, 10, and 14 post-infection (PI) on relative bacterial abundance (%) at the genus level in the (<b>A</b>) ileal lumen (IL-C) and (<b>B</b>) ileal mucosa (IL-M). Non-infected birds = C, infected birds = IF.</p>
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<p>Effect of <span class="html-italic">Eimeria maxima</span> on differentially abundant bacterial genera as determined by linear discriminant analysis (LDA) effect size (LEfSe) analysis in jejunal lumen (J-C, (<b>A</b>)) and mucosal (J-M, (<b>B</b>)) bacterial populations. C—uninfected chickens, IF—infected chickens.</p>
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<p>Effect of <span class="html-italic">Eimeria maxima</span> on differentially abundant bacterial genera as determined by linear discriminant analysis (LDA) effect size (LEfSe) analysis in ileal lumen (IL-C, (<b>A</b>)) and mucosal (IL-M, (<b>B</b>)) bacterial populations. C—uninfected chickens, IF—infected chickens.</p>
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<p>Effect of <span class="html-italic">Eimeria maxima</span> infection on mean proportion (%) of predicted MetaCyc pathways (up to top 21 pathways shown) in the jejunal luminal (J-C) (<b>A</b>) and jejunal mucosal (J-M) (<b>B</b>) populations.</p>
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<p>Effect of <span class="html-italic">Eimeria maxima</span> infection on the mean proportion (%) of predicted MetaCyc pathways (up to top 21 pathways shown) in the ileal luminal (IL-C) (<b>A</b>) and ileal mucosal (IL-M) (<b>B</b>) populations.</p>
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10 pages, 238 KiB  
Article
Drinking Water Quality Management for Broiler Performance and Carcass Characteristics
by Naser Amir Ebrahimi, Ali Nobakht, Hakan İnci, Valiollah Palangi, Marian Suplata and Maximilian Lackner
World 2024, 5(4), 952-961; https://doi.org/10.3390/world5040048 (registering DOI) - 15 Oct 2024
Viewed by 247
Abstract
Objective: This study aimed to assess the impact of water quality as determined by its physical, chemical, and biological composition collected from five distinct points in Maragheh, Iran, on the performance and carcass traits of Ross-308 commercial broilers (mix of male and female) [...] Read more.
Objective: This study aimed to assess the impact of water quality as determined by its physical, chemical, and biological composition collected from five distinct points in Maragheh, Iran, on the performance and carcass traits of Ross-308 commercial broilers (mix of male and female) during the grower (11–24 days) and finisher (25–42 days) periods. Materials and methods: A total of 240 broilers were involved in the study, divided into five treatments with four replicates and 12 birds per replicate. In this study, a randomized design was used. Water samples were collected from five different points, and broilers were provided with these water sources during the grower and finisher periods. Water samples for testing were prepared from the water wells of the meat poultry farms located in the northern, eastern, western, and southern lands, and the experimental farm, using hygienic and scientific methods. Performance parameters, including body weight, feed conversion ratio (FCR), and water intake, were measured. Results: During the grower period, no significant effects on performance and water intake were observed across the different water sources (p > 0.05). However, in the finisher period, significant differences were noted (p ≥ 0.05). The use of water from point A (control group) led to reduced water consumption, body weight, and increased FCR (p < 0.05). The northern water source exhibited the optimum FCR during the finisher period (p < 0.05). Throughout the entire experimental period, the water source significantly influenced broiler performance, with the northern water source (point B) corresponding with the highest weight gain and production index with the least feed intake (p < 0.05). Despite these variations, no significant changes were observed in the broilers’ carcass traits across different water sources (p ≥ 0.05). Conclusions: In conclusion, the study revealed that various drinking water sources, while not significantly impacting carcass quality traits, exerted notable effects on broilers’ performance. The northern water source emerged as particularly favorable, demonstrating superior weight gain and a production index with efficient feed utilization. These findings underscore the importance of water quality in poultry management, particularly during the finisher period, and highlight its potential influence on broiler performance. Full article
14 pages, 8195 KiB  
Article
The Application of Duck Embryonic Fibroblasts CCL-141 as a Cell Model for Adipogenesis
by Dan-Dan Sun, Xiao-Qin Li, Yong-Tong Liu, Meng-Qi Ge and Zhuo-Cheng Hou
Animals 2024, 14(20), 2973; https://doi.org/10.3390/ani14202973 (registering DOI) - 15 Oct 2024
Viewed by 281
Abstract
The duck embryo fibroblast cell line CCL-141, which is currently the only commercialized duck cell line, has been underexplored in adipogenesis research. (1) Background: This study establishes an experimental protocol to induce adipogenesis in CCL-141 cells, addressing the importance of understanding gene functions [...] Read more.
The duck embryo fibroblast cell line CCL-141, which is currently the only commercialized duck cell line, has been underexplored in adipogenesis research. (1) Background: This study establishes an experimental protocol to induce adipogenesis in CCL-141 cells, addressing the importance of understanding gene functions in this process. (2) Methods: Chicken serum, fatty acids, insulin, and all-trans retinoic acid were used to treat CCL-141 cells, with adipogenesis confirmed by Oil Red O staining and gene expression quantification. CRISPR/Cas9 technology was applied to knockout PPARγ, and the resulting adipogenic phenotype was assessed. (3) Results: The treatments promoted adipogenesis, and the knockout of PPARγ validated the cell line’s utility for gene function studies. (4) Conclusions: CCL-141 cells are a suitable model for investigating duck adipogenesis, contributing to the understanding of regulatory factors in this biological process. Full article
(This article belongs to the Section Animal Genetics and Genomics)
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<p>Establishment of duck <span class="html-italic">PPARγ</span>-knockout pooled cells using CRISPR/Cas9 system. (<b>A</b>) Structure of <span class="html-italic">PPARγ</span> and location of target sequence. (<b>B</b>) Analysis of knockout efficiency using website analysis based on Sanger sequencing peak graph. (<b>C</b>) Analysis of <span class="html-italic">PPARγ</span> knockout efficiency using single cloning. Red font corresponds to target sequence. Deleted nucleotides are marked with dashes, inserted nucleotides are represented with caret “^”, mutational nucleotides are represented with lowercase letters, and protospacer adjacent motif (PAM) sequence is indicated with italics and underlined.</p>
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<p>Inducing effects of different culture medium components on CCL-141 cells. (<b>A</b>) Representative images of Oil Red O staining after 72 h of induction for different culture groups: (a) EMEM with 10% FBS as control; (b) EMEM with 10% chicken serum; (c) EMEM with 10% CS, 1:100 fatty acids, and 10 ug/mL insulin; and (d) EMEM with 10% CS, 1:100 fatty acids, 10 ug/mL insulin, and 40 ug/mL all-trans retinoic acid. (<b>B</b>) Comparison of lipid droplet content in different groups extracted after Oil Red O staining (different lowercase letters on columns indicate significant differences, <span class="html-italic">p</span> &lt; 0.05).</p>
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<p>Differential expression of marker genes for adipogenesis in differentiating groups induced by culture medium containing different components for 48 h and 72 h (different lowercase letters indicate significant differences, <span class="html-italic">p</span> &lt; 0.05).</p>
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<p>Differential expression of marker genes for adipogenesis in wild-type and knockout groups before and after 72 h of induction (different lowercase letters indicate significant differences, <span class="html-italic">p</span> &lt; 0.05).</p>
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<p>Differences in protein expression level and lipid deposition in wild-type (WT) and <span class="html-italic">PPARγ</span>-KO pooled cells. (<b>A</b>) Western blotting analysis of CCL-141. Protein samples of WT and <span class="html-italic">PPARγ</span>-KO pooled cells were extracted and Western blot analysis was performed against <span class="html-italic">PPARγ</span> antibody as per procedure described in “Materials and Methods” section. (<b>B</b>) Gray value analysis of protein expression level in wild-type and <span class="html-italic">PPARγ</span>-KO pooled cells (** <span class="html-italic">p</span> &lt; 0.01). (<b>C</b>) Representative images of Oil Red O staining after 72 h of induction in wild-type and knockout groups. (<b>D</b>) Comparison of lipid droplet content in different groups extracted after Oil Red O staining (different lowercase letters indicate significant differences, <span class="html-italic">p</span> &lt; 0.05).</p>
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<p>Cell proliferation curves of wild-type and knockout groups at different time points (** <span class="html-italic">p</span> &lt; 0.01).</p>
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19 pages, 1558 KiB  
Article
Genome of Russian Snow-White Chicken Reveals Genetic Features Associated with Adaptations to Cold and Diseases
by Ivan S. Yevshin, Elena I. Shagimardanova, Anna S. Ryabova, Sergey S. Pintus, Fedor A. Kolpakov and Oleg A. Gusev
Int. J. Mol. Sci. 2024, 25(20), 11066; https://doi.org/10.3390/ijms252011066 (registering DOI) - 15 Oct 2024
Viewed by 255
Abstract
Russian Snow White (RSW) chickens are characterized by high egg production, extreme resistance to low temperatures, disease resistance, and by the snow-white color of the day-old chicks. Studying the genome of this unique chicken breed will reveal its evolutionary history and help to [...] Read more.
Russian Snow White (RSW) chickens are characterized by high egg production, extreme resistance to low temperatures, disease resistance, and by the snow-white color of the day-old chicks. Studying the genome of this unique chicken breed will reveal its evolutionary history and help to understand the molecular genetic mechanisms underlying the unique characteristics of this breed, which will open new breeding opportunities and support future studies. We have sequenced and made a de novo assembly of the whole RSW genome using deep sequencing (250×) by the short reads. The genome consists of 40 chromosomes with a total length of 1.1 billion nucleotide pairs. Phylogenetic analysis placed the RSW near the White Leghorn, Fayoumi, and Houdan breeds. Comparison with other chicken breeds revealed a wide pool of mutations unique to the RSW. The functional annotation of these mutations showed the adaptation of genes associated with the development of the nervous system, thermoreceptors, purine receptors, and the TGF-beta pathway, probably caused by selection for low temperatures. We also found adaptation of the immune system genes, likely driven by selection for resistance to viral diseases. Integration with previous genome-wide association studies (GWAS) suggested several causal single nucleotide polymorphisms (SNPs). Specifically, we identified an RSW-specific missense mutation in the RALYL gene, presumably causing the snow-white color of the day-old chicks, and an RSW-specific missense mutation in the TLL1 gene, presumably affecting the egg weight. Full article
(This article belongs to the Special Issue Molecular Research in Avian Genetics)
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<p>The BUSCO assessment results for the 4 genome assemblies, showing the percentages and categories of the single-copy orthologs from the aves_odb10 data set (total genes = 8338) in each genome assembly: GGRsw1—the genome assembly of the RSW in this study; GGswu—the genome assembly of the Huxu breed [<a href="#B9-ijms-25-11066" class="html-bibr">9</a>]; GRCg6a—the genome assembly of the Red Junglefowl (official reference genome from the Genome Reference Consortium); and GRCg7b—the genome assembly of the broiler (official reference genome from the Genome Reference Consortium). GGRSw1 contains a greater number of single-copy orthologs, and lower numbers of missing and fragmented genes.</p>
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<p>A phylogenetic tree based on the comparison of the whole genome sequences of chickens of different breeds. Red—The Red Junglefowl is a chicken from Southeast Asia, from which domestic chickens probably originate; blue—“European” breeds; purple—American breeds; green—broiler breeds; yellow—Chinese breeds from Yunnan province; Brown—Chinese breeds not of Yunnan origin; and gray—other breeds.</p>
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<p>(<b>A</b>). The genomic regions unique to the Russian Snow White longer than 1000 bp. A map of all GGRsw1 chromosomes is shown, with green bars marking the genomic regions unique to the Russian White breed. (<b>B</b>). The figure shows, for each RSW-specific sequence, what proportion of that sequence is composed of G4s and tandem repeats: X axis—the fraction of G4s; Y axis—the fraction of tandem repeats.</p>
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<p>(<b>A</b>). The genomic regions unique to the Russian Snow White longer than 1000 bp. A map of all GGRsw1 chromosomes is shown, with green bars marking the genomic regions unique to the Russian White breed. (<b>B</b>). The figure shows, for each RSW-specific sequence, what proportion of that sequence is composed of G4s and tandem repeats: X axis—the fraction of G4s; Y axis—the fraction of tandem repeats.</p>
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12 pages, 1262 KiB  
Article
Evaluation of the Efficacy of Three Newcastle Disease Vaccines Produced at the National Veterinary Institute, Bishoftu, Ethiopia, at Different Temperature Storage Conditions
by Teferi Degefa, Mahlet Birehanu, Demise Mulugeta, Henok Ferede, Endalkachew Girma, Anberber Alemu, Dassalegn Muleta, Abebe Mengesha Aga, Debebe Shimeket, Dereje Nigussie Woldemichael, Mirtneh Akalu and Fanos Tadesse Woldemariyam
Acta Microbiol. Hell. 2024, 69(4), 212-223; https://doi.org/10.3390/amh69040020 (registering DOI) - 15 Oct 2024
Viewed by 239
Abstract
Newcastle disease, which affects poultry and is endemic in many nations across the world, is caused by Avian Paramyxovirus-1 (APMV-1). This experimental study was conducted from January to June 2021 at the National Veterinary Institute (NVI) to evaluate the virus viability and antibody [...] Read more.
Newcastle disease, which affects poultry and is endemic in many nations across the world, is caused by Avian Paramyxovirus-1 (APMV-1). This experimental study was conducted from January to June 2021 at the National Veterinary Institute (NVI) to evaluate the virus viability and antibody titer of Newcastle disease vaccines (Hichner’s B1, Lasota, and ThermostableI2) stored at different temperature storage conditions. Chickens (12 treatment groups and 1 control group) were vaccinated and challenged with the virulent ND virus (0.5 × 106.5 embryonic lethal dose fifty (ELD50)). The immune responses (antibody titers) of chickens were evaluated using hemagglutination (HA) and hemagglutination inhibition (HI) assays. The Newcastle disease vaccines (Hachiner’s B1 (ND-HB1), ND-Lasota, and ND-Thermostable I2) stored at +4 °C HI-induced antibody titers of 151 (±103.3), 136 (±53.4), and 145 (±91) on day 14, respectively, whereas on day 21, they increased to 160 (±82) for ND-HB1 and 144 (±74.5) for ND-Lasota. ND-Thermostable I2 showed a decrement to 133 (±44.8). All three vaccines stored at different temperature storage conditions (+4, +23, and +30 °C) used in this experiment induced antibody titers greater than 128 on day 28 post-vaccination, except the Newcastle disease vaccine Thermostable I2 stored at +30 °C. The vaccines collected from private veterinary drugstores (customer vaccines Hachiner’s B1 and ND-Thermostable I2) used in this experiment induced very low antibody titers, less than 128 antibody titers, from days 14 to 21. Statistically significant induced mean antibody titers were observed for chickens that received vaccines stored at different temperature storage conditions for 72 h (p < 0.05), except for the ND-HB1 mean HI-induced antibody titer at days 7 and 28. Further, vaccine protection was confirmed by inoculation of both the vaccinated (treatment groups) and control groups by the virulent ND virus, where the control group started dying three days post-challenge but all chicks that received the vaccines survived. Overall, this study showed the impact of temperature storage conditions on the antibody titer and their effect on the titer of the viable virus in the vaccine, and thereby its protective capacity, warranting appropriate cold chain management of the vaccines along the value chain. Full article
(This article belongs to the Special Issue Feature Papers in Medical Microbiology in 2024)
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<p>Induced antibody titer of the ND-Lasota vaccine from day zero up to twenty-eight days; 4 (blue), 23 (yellow), 30 (gray), and customer vaccine (bright yellow) indicate the temperature storage conditions of the vaccines.</p>
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<p>Induced antibody titers of ND-HB1 vaccine from day zero up to twenty-eight; 4 (blue), 23 (yellow), and 30 (gray) indicate the temperature storage conditions of the vaccines.</p>
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<p>Induced antibody titers of Newcastle Thermostable I2 vaccine from day zero up to twenty-eight days; 4 (blue), 23 (yellow), 30 (gray), and customer vaccine (bright yellow) indicate the temperature storage conditions of the vaccines.</p>
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<p>Gel electrophoresis of the virulent strain of Newcastle disease virus was detected using polymerase chain reaction (PCR). Legend: S1–S4: brain tissue from control chickens that died after challenge; S5, S7 and S9: trachea swabs from control chickens that died after the challenge; S6 and S8: spleen samples from vaccinated and challenged chickens.</p>
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16 pages, 769 KiB  
Article
Expression of Genes Related to Meat Productivity, Metabolic and Morphological Significance of Broiler Chickens with the Use of Nutritional Phytochemicals
by Marina I. Selionova, Vladimir I. Trukhachev, Artem Yu. Zagarin, Egor I. Kulikov, Dmitry M. Dmitrenko, Vera N. Martynova, Arina K. Kravchenko and Vladimir G. Vertiprakhov
Animals 2024, 14(20), 2958; https://doi.org/10.3390/ani14202958 - 14 Oct 2024
Viewed by 240
Abstract
The study aimed to analyze gene expression linked to skeletal muscle growth and lipid metabolism in broiler chickens fed with plant extracts. Five groups of chickens were formed: four experimental groups and one control group. The diets of the experimental groups were supplemented [...] Read more.
The study aimed to analyze gene expression linked to skeletal muscle growth and lipid metabolism in broiler chickens fed with plant extracts. Five groups of chickens were formed: four experimental groups and one control group. The diets of the experimental groups were supplemented with different plant extracts: chicory, St. John’s wort, maral root, and creeping thyme, whereas the control group received feed without phytobiotic compounds. Weekly weighings were conducted (n = 36). The chickens were slaughtered at day 26 for tissue sampling of four birds from each group. Gene expression (MYOG, MSTN, FASN) related to muscle growth and fatty acid synthesis was analyzed using the β-actin ACTB gene as a reference. Blood samples were taken at day 35 for biochemical analysis and anatomical dissection was performed. The study revealed that using plant extracts from chicory, thyme, and maral root increased MYOG gene activity by 4.21, 7.45, and 8.93 times, respectively. T. serpyllum extract boosted the MSTN gene by 10.93 times, impacting muscle growth regulation. FASN gene expression for fatty acid synthesis increased significantly by 18.22–184.12 times with plant extracts. The best results regarding meat productivity of chickens were obtained when using R. carthamoides extract. The results of the study will serve as a basis for further development of a phytocomposition designed to increase the meat productivity of broiler chickens in the production of environmentally safe poultry products. Full article
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<p>Relative quantification (RQ) of expression level of genes related to the growth and development of skeletal muscle tissues in breast muscles of broiler chickens (<span class="html-italic">Gallus gallus</span> L.) of the “Smena 9” cross when using phytobiotics in the diet: (RQ—fold change in expression level compared to the first group, where the parameter was taken as 1; *, **—the difference in ΔCt values is statistically significant compared to the control group at <span class="html-italic">p</span> ≤ 0.05, <span class="html-italic">p</span> ≤ 0.01 according to the t-criterion; the horizontal dashed line indicates the level of gene expression in the control group; results are presented as the mean with the standard error of the mean (M ± SEM) for mRNA expression).</p>
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<p>Relative quantification (RQ) of expression level of the <span class="html-italic">FASN</span> gene in the breast muscle tissues of broiler chickens (<span class="html-italic">Gallus gallus</span> L.) of the “Smena 9” cross when using phytobiotics in the diet: (RQ—fold change in expression level compared to the first group, where the parameter was taken as 1; *, **—the difference in ΔCt values is statistically significant compared to the control group at <span class="html-italic">p</span> ≤ 0.01, <span class="html-italic">p</span> ≤ 0.001 according to the t-criterion; results are presented as the mean with the standard error of the mean (M ± SEM) for mRNA expression).</p>
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24 pages, 5046 KiB  
Article
Ultrasensitive Electrochemical Detection of Salmonella typhimurium in Food Matrices Using Surface-Modified Bacterial Cellulose with Immobilized Phage Particles
by Wajid Hussain, Huan Wang, Xiaohan Yang, Muhammad Wajid Ullah, Jawad Hussain, Najeeb Ullah, Mazhar Ul-Islam, Mohamed F. Awad and Shenqi Wang
Biosensors 2024, 14(10), 500; https://doi.org/10.3390/bios14100500 - 14 Oct 2024
Viewed by 548
Abstract
The rapid and sensitive detection of Salmonella typhimurium in food matrices is crucial for ensuring food safety. This study presents the development of an ultrasensitive electrochemical biosensor using surface-modified bacterial cellulose (BC) integrated with polypyrrole (Ppy) and reduced graphene oxide (RGO), further functionalized [...] Read more.
The rapid and sensitive detection of Salmonella typhimurium in food matrices is crucial for ensuring food safety. This study presents the development of an ultrasensitive electrochemical biosensor using surface-modified bacterial cellulose (BC) integrated with polypyrrole (Ppy) and reduced graphene oxide (RGO), further functionalized with immobilized S. typhimurium-specific phage particles. The BC substrate, with its ultra-fibrous and porous structure, was modified through in situ oxidative polymerization of Ppy and RGO, resulting in a highly conductive and flexible biointerface. The immobilization of phages onto this composite was facilitated by electrostatic interactions between the polycationic Ppy and the negatively charged phage capsid heads, optimizing phage orientation and enhancing bacterial capture efficiency. Morphological and chemical characterization confirmed the successful fabrication and phage immobilization. The biosensor demonstrated a detection limit of 1 CFU/mL for S. typhimurium in phosphate-buffered saline (PBS), with a linear detection range spanning 100 to 107 CFU/mL. In real samples, the sensor achieved detection limits of 5 CFU/mL in milk and 3 CFU/mL in chicken, with a linear detection range spanning 100 to 106 CFU/mL, maintaining high accuracy and reproducibility. The biosensor also effectively discriminated between live and dead bacterial cells, demonstrating its potential in real-world food safety applications. The biosensor performed excellently over a wide pH range (4–10) and remained stable for up to six weeks. Overall, the developed BC/Ppy/RGO–phage biosensor offers a promising tool for the rapid, sensitive, and selective detection of S. typhimurium, with robust performance across different food matrices. Full article
(This article belongs to the Special Issue Advancements in Biosensors for Foodborne Pathogens Detection)
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<p>Fabrication of BC/Ppy/RGO composite. Immobilization of the <span class="html-italic">S. typhimurium</span>-specific phages to develop a BC/Ppy/RGO-phage biointerface for the electrochemical detection of <span class="html-italic">S. typhi</span> in milk and chicken samples using DPV.</p>
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<p>FE-SEM investigation of pristine BC before and after modification with Ppy, RGO, and immobilized phages. (<b>A</b>,<b>B</b>) Pristine BC at different magnifications, (<b>C</b>) Ppy polymerization on BC, (<b>D</b>,<b>E</b>) BC/Ppy/RGO at different magnifications, and (<b>F</b>,<b>G</b>) show the immobalized phages on BC/Ppy/RGO biointerface and red color arrows represent the individual phage particles. (<b>H</b>) Magnified image of the phage particles attached to the BC/Ppy/RGO shown in rectangular and the yellow circles represent individual phage particle. Elemental mapping images of BC/Ppy/RGO (<b>I</b>), carbon (<b>J</b>), oxygen (<b>K</b>), and nitrogen (<b>L</b>).</p>
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<p>Characterization of modified BC composites and BC-phage biointerfaces. (<b>A</b>) XRD patterns of GO, RGO, BC, Ppy, BC/Ppy, and BC/Ppy/RGO and physiological changes of GO in RGO; (<b>B</b>) FT-IR spectra of BC, BC/Ppy, BC/Ppy/RGO, and BC/Ppy/RGO-phage; (<b>C</b>) Selected magnified area in (<b>B</b>) FT-IR-fingerprint of BC/Ppy, BC/Ppy/RGO, and BC/Ppy/RGO-phage ranging from 1800 to 800 cm<sup>−1</sup>. (<b>D</b>) XPS wide-scan patterns of BC, BC/Ppy, BC/Ppy/RGO, and BC/Ppy/RGO–phage. (<b>E</b>) N 1s core-level spectra of BC/Ppy/RGO-phage biointerface.</p>
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<p>Plaque assays for anti-<span class="html-italic">Salmonella</span> potential of BC-based biointerfaces. (<b>A</b>–<b>E</b>) Plaque formation of materials such as BC, BC/Ppy, and BC/Ppy/RGO (I–III). (<b>F</b>–<b>J</b>) Plaque formation after immobilization of phages on different BC-based bio-interfaces such as BC, BC/Ppy, and BC/Ppy/RGO (I–III)–phage. (<b>K</b>–<b>O</b>) Sonicated interfaces with immobilized phages and their lytic activity.</p>
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<p>Infectious dynamics of phages immobilized on surface-modified BC against <span class="html-italic">S. typhimuruim</span> and their density under confocal microscopy. (<b>A</b>) Growth reduction curves in terms of optical density (OD<sub>600</sub>) of <span class="html-italic">S. typhi</span>, with free phages and phages immobilized on BC, BC/Ppy, BC/Ppy/RGO (I-III), (<b>B</b>) pristine BC, and (<b>C</b>) immobilized phages on BC (in-focus). (<b>D</b>–<b>F</b>) Density of stained phage particles (in-focus), while the composite is out of focus; (<b>D</b>) immobilized phages on pristine BC, (<b>E</b>) immobilized phages on BC/Ppy, and (<b>F</b>) immobilized phages on BC/Ppy/RGO.</p>
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<p>Electrochemical characterization of the surface BC-based modified electrodes for <span class="html-italic">S. typhi</span> detection. CV curves of different BC and BC-modified electrodes in cyclic voltammograms of the Fe(CN)<sub>6</sub><sup>3−</sup>/Fe(CN)<sub>6</sub><sup>4−</sup> redox system, such as BC/Ppy, and different concentrations of RGO and immobilized phages, including (<b>A</b>) BC/Ppy/RGO (I)-phage, (<b>B</b>) BC/Ppy/RGO (II)-phage, and (<b>C</b>) BC/Ppy/RGO (III)-phage. DPV-based current responses of different concentrations of RGO with Ppy and phages (<b>D</b>) BC/Ppy/RGO (I)-phage, (<b>E</b>) BC/Ppy/RGO (II)-phage, and (<b>F</b>) BC/Ppy/RGO (III)-phage.</p>
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<p>Specificity, live/dead cell discrimination, and practical applications of the BC/Ppy/RGO-phage biosensor for electrochemical detection of <span class="html-italic">S. typhi</span>. (<b>A</b>) DPV current response towards detected <span class="html-italic">S. typhi</span> in PBS, (<b>B</b>) linear range of <span class="html-italic">S. typhi</span> detection in PBS, (<b>C</b>) specificity of the biosensor, (<b>D</b>) biosensor discrimination for live, dead, and mixture of live/dead <span class="html-italic">S. typhi</span>, (<b>E</b>) DPV curves of the detected <span class="html-italic">S. typhi</span> in milk, (<b>F</b>) linear range of <span class="html-italic">S. typhi</span> in milk, (<b>G</b>) detection of <span class="html-italic">S. typhi</span> in chicken samples at different concentrations, and (<b>H</b>) linear range of detection in chicken. The standard deviations of triplicate analyses for each experiment are indicated by error bars.</p>
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19 pages, 4147 KiB  
Article
Research on Section Coal Pillar Deformation Prediction Based on Fiber Optic Sensing Monitoring and Machine Learning Algorithms
by Dingding Zhang, Yu Wang, Jianfeng Yang, Dengyan Gao and Jing Chai
Appl. Sci. 2024, 14(20), 9347; https://doi.org/10.3390/app14209347 (registering DOI) - 14 Oct 2024
Viewed by 273
Abstract
The mining face under the close coal seam group is affected by the superposition of the concentrated stress of the overlying residual diagonally intersecting coal pillar and the mining stress, which can easily cause the instability and damage of the section coal pillars [...] Read more.
The mining face under the close coal seam group is affected by the superposition of the concentrated stress of the overlying residual diagonally intersecting coal pillar and the mining stress, which can easily cause the instability and damage of the section coal pillars during the process of mining back to the downward face. Additionally, the traditional methods of monitoring such as numerical simulation, drilling peeping, and acoustic emission fail to realize the real-time and accurate deformation monitoring of the internal deformation of the section coal pillars. The introduction of the drill-hole-implanted fiber-optic grating monitoring method can realize real-time deformation monitoring for the whole area inside the coal pillar, which solves the short board problem of coal pillar deformation monitoring. However, fiber-optic monitoring is easily disturbed by the external environment, which is especially sensitive to the background noise of the complex underground mining environment. Therefore, taking the live chicken and rabbit well of Shaanxi Daliuta Coal Mine as the engineering background, the ensemble empirical modal decomposition (EEMD) is introduced for primary noise reduction and signal reconstruction by the threshold determination (DE) algorithm, and then the singular matrix decomposition (SVD) is introduced for secondary noise reduction. Finally, a machine learning algorithm is combined with the noise reduction algorithm for the prediction of the fiber grating strain signals of coal pillar in a zone, and DBO-LSTM-BP is constructed as the prediction model. The experimental results demonstrate that compared with the other two noise reduction prediction models, the SNR of the EEMD-DE-SVD-DBO-LSTM-BP model is improved by 0.8–2.3 dB on average, and the prediction accuracy is in the range of 88–99%, which realizes the over-advanced prediction of the deformation state of the coal column in the section. Full article
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<p>Working face layout and section coal pillar setup in the study area.</p>
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<p>Coal bed map.</p>
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<p>Fiber-optic grating monitoring area.</p>
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<p>Fiber-optic grating strain monitoring point tendency profiles.</p>
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<p>Fiber-optic grating string monitoring point arrangement.</p>
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<p>Results of strain monitoring of 5 fiber grating boreholes at measurement points 1–9. (<b>a</b>) Hole 1. (<b>b</b>) Hole 2. (<b>c</b>) Hole 3. (<b>d</b>) Hole 4. (<b>e</b>) Hole 5.</p>
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<p>Results of strain monitoring of 5 fiber grating boreholes at measurement points 1–9. (<b>a</b>) Hole 1. (<b>b</b>) Hole 2. (<b>c</b>) Hole 3. (<b>d</b>) Hole 4. (<b>e</b>) Hole 5.</p>
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<p>EEMD denoising decomposition of the signal from borehole 1, measurement point 8.</p>
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<p>Dispersion entropy of IMF components for borehole 1, measurement point 8.</p>
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<p>Dispersion entropy ratio of IMF components for borehole 1, measurement point 8.</p>
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<p>LSTM neural network graph.</p>
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<p>Neural network hierarchy diagram.</p>
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<p>Structure of the constructed noise reduction prediction model.</p>
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<p>EEMD-DE-SVD-DBO-LSTM-BP strain prediction results. (<b>a</b>) Test Set 1. (<b>b</b>) Test Set 2. (<b>c</b>) Test Set 3. (<b>d</b>) Test Set 4.</p>
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<p>EEMD-DE-SVD-DBO-LSTM-BP strain prediction results. (<b>a</b>) Test Set 1. (<b>b</b>) Test Set 2. (<b>c</b>) Test Set 3. (<b>d</b>) Test Set 4.</p>
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19 pages, 4042 KiB  
Article
Comparative Study of an Antioxidant Compound and Ethoxyquin on Feed Oxidative Stability and on Performance, Antioxidant Capacity, and Intestinal Health in Starter Broiler Chickens
by Yong Xiao, Xuyang Gao and Jianmin Yuan
Antioxidants 2024, 13(10), 1229; https://doi.org/10.3390/antiox13101229 - 13 Oct 2024
Viewed by 870
Abstract
Concerns over the safety of ethoxyquin (EQ) highlight the need for safer, more effective feed antioxidants. This study investigated a healthier antioxidant compound (AC) as a potential alternative to EQ in broilers. A total of 351 one-day-old Arbor Acres Plus male broilers were [...] Read more.
Concerns over the safety of ethoxyquin (EQ) highlight the need for safer, more effective feed antioxidants. This study investigated a healthier antioxidant compound (AC) as a potential alternative to EQ in broilers. A total of 351 one-day-old Arbor Acres Plus male broilers were randomly assigned to three treatments for 21 days: control (CON), EQ group (200 g/ton EQ at 60% purity), and AC group (200 g/ton AC containing 18% butylated hydroxytoluene, 3% citric acid, and 1% tertiary butylhydroquinone). AC supplementation reduced the acid value, peroxide value, and malondialdehyde content in stored feed, decreased feed intake and the feed conversion ratio without affecting body weight gain, and enhanced antioxidant capacity (liver total antioxidant capacity and superoxide dismutase; intestinal catalase and glutathione peroxidase 7). It improved intestinal morphology and decreased barrier permeability (lower diamine oxidase and D-lactate), potentially by promoting ZO-1, Occludin, and Mucin2 expression. The AC also upregulated NF-κB p50 and its inhibitor (NF-κB p105), enhancing immune regulation. Additionally, the AC tended to increase beneficial gut microbiota, including Lactobacillus, and reduced Bacteroides, Corprococcus, and Anaeroplasma. Compared to EQ, the AC further enhanced feed oxidative stability, the feed conversion ratio, intestinal morphology and barrier functions, and inflammatory status, suggesting its potential as a superior alternative to EQ for broiler diets. Full article
(This article belongs to the Special Issue Oxidative Stress in Poultry Reproduction and Nutrition)
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<p>Oxidative stability of feeds after three and six weeks of storage. N = 3. (<b>A</b>) Acid value (AV); (<b>B</b>) peroxide value (POV); (<b>C</b>) malondialdehyde (MDA) content. CON, basal diet; EQ, basal diet + 200 g/ton ethoxyquin; AC, basal diet + 200 g/ton antioxidant compound. <sup>abc</sup> Different superscript letters indicate significant differences (<span class="html-italic">p</span> &lt; 0.05).</p>
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<p>Growth performance of broilers. N = 9. (<b>A</b>) Body weight gain (BWG); (<b>B</b>) feed intake (FI); (<b>C</b>) feed conversion ratio (FCR). CON, basal diet; EQ, basal diet + 200 g/ton ethoxyquin; AC, basal diet + 200 g/ton antioxidant compound. <sup>ab</sup> Different superscript letters indicate significant differences (<span class="html-italic">p</span> &lt; 0.05).</p>
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<p>Liver antioxidant capacity of broilers. N = 8. (<b>A</b>) Total antioxidant capacity (T-AOC); (<b>B</b>) superoxide (SOD) activity; (<b>C</b>) catalase (CAT) activity; (<b>D</b>) glutathione peroxidase (GSH-Px) activity; (<b>E</b>) reduced glutathione (GSH) content; (<b>F</b>) malondialdehyde (MDA) content. CON, basal diet; EQ, basal diet + 200 g/ton ethoxyquin; AC, basal diet + 200 g/ton antioxidant compound. <sup>ab</sup> Different superscript letters indicate significant differences (<span class="html-italic">p</span> &lt; 0.05).</p>
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<p>Intestinal barrier permeability and intestinal-barrier-related protein expression. N = 8. (<b>A</b>) Serum diamine oxidase activity and (<b>B</b>) D-lactate contents. (<b>C</b>–<b>F</b>) The relative protein expression of ZO-1, Occludin, and MUC2 in the jejunum. CON, basal diet; EQ, basal diet + 200 g/ton ethoxyquin; AC, basal diet + 200 g/ton antioxidant compound. Abbreviations: DAO, diamine oxidase. <sup>ab</sup> Different superscript letters indicate significant differences (<span class="html-italic">p</span> &lt; 0.05).</p>
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<p>Intestinal-antioxidant-related protein expression. N = 8. (<b>A</b>–<b>D</b>) Relative protein levels of kelch-like ECH-associated protein 1 (KEAP1), nuclear factor erythroid 2-related factor 2 (NRF2), and catalase (CAT) in jejunum. (<b>E</b>–<b>H</b>) Relative protein levels of heme oxygenase-1 (HO-1), glutathione peroxidase 7 (GPX7), and NAD(P)H quinone dehydrogenase 1 (NQO1) in jejunum. N = 8. CON, basal diet; CON, basal diet; EQ, basal diet + 200 g/ton ethoxyquin; AC, basal diet + 200 g/ton antioxidant compound. <sup>ab</sup> Different superscript letters indicate significant differences (<span class="html-italic">p</span> &lt; 0.05).</p>
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<p>Expression of inflammatory cytokines at gene level and protein level. N = 8. (<b>A</b>) Relative mRNA expression of <span class="html-italic">IL-1β</span>, <span class="html-italic">IL-18</span>, <span class="html-italic">IL-6</span>, <span class="html-italic">IFN-γ</span>, and <span class="html-italic">TNF-α</span> in jejunum. (<b>B</b>–<b>F</b>) Relative protein levels of NF-κB p50, NF-κB p105, inhibitor of nuclear factor kappa-B (IκB), and toll-like receptor 4 (TLR4) in jejunum. CON, basal diet; EQ, basal diet + 200 g/ton ethoxyquin; AC, basal diet + 200 g/ton antioxidant compound. <sup>ab</sup> Different superscript letters indicate significant differences (<span class="html-italic">p</span> &lt; 0.05).</p>
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<p>Changes in microbial diversity in the cecum of broilers. N = 6. (<b>A</b>) Venn diagram of the operational taxonomic units (OTUs) among all groups. (<b>B</b>) Bacterial richness (Chao1 and Observed species) and diversity (Shannon and Simpson) were evaluated by Kruskal–Wallis rank sum test and Dunn’s test. (<b>C</b>) The rarefaction curve shows the Chao1 index of each group under the same sampling depth. (<b>D</b>) The principal coordinate analysis (PCoA) and (<b>E</b>) non-metric multidimensional scaling (NMDS) analysis were conducted at the ASV level (the distance matrix was analyzed for statistical significance using PERMANOVA). CON, basal diet; EQ, basal diet + 200 g/ton ethoxyquin; AC, basal diet + 200 g/ton compound antioxidant.</p>
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<p>Changes in microbial community composition in the cecum of broilers. N = 6. (<b>A</b>) Distribution of cecal microbiota at the phylum level. (<b>B</b>) Heatmap showing the relative abundance of cecal microbiota at the phylum level. (<b>C</b>–<b>E</b>) Bacteria with differences in relative abundance among the top 10 phyla, including Firmicutes, Bacteroidetes, and the ratio of Bacteroidetes and Firmicutes. (<b>F</b>) Distribution of cecal microbiota at the genus level. (<b>G</b>) Heatmap showing the relative abundance of cecal microbiota at the genus level. (<b>H</b>–<b>K</b>) Bacteria with differences in relative abundance among the top 20 genera, including <span class="html-italic">Lactobacillus</span>, <span class="html-italic">Bacteroides</span>, <span class="html-italic">Coprococcus</span>, and <span class="html-italic">Anaeroplasma</span>. Differences in microbial abundance between the CON and AC groups were analyzed using the Wilcoxon rank sum test. CON, basal diet; EQ, basal diet + 200 g/ton ethoxyquin; AC, basal diet + 200 g/ton antioxidant compound.</p>
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<p>Graphic summary of the effects of the antioxidant compound on feed antioxidant protection and broiler health. Antioxidant compound: including 18% butylated hydroxytoluene, 3% citric acid, and 1% tertiary butylhydroquinone.</p>
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17 pages, 2324 KiB  
Article
Local Inflammatory and Systemic Antibody Responses Initiated by a First Intradermal Administration of Autogenous Salmonella-Killed Vaccines and Their Components in Pullets
by Jossie M. Santamaria, Chrysta N. Beck and Gisela F. Erf
Vaccines 2024, 12(10), 1159; https://doi.org/10.3390/vaccines12101159 - 11 Oct 2024
Viewed by 424
Abstract
Vaccination strategies are used to manage Salmonella in chickens. Salmonella-killed vaccines are considered safer since they are inactivated. However, little is known regarding the cellular immune activities at the site of vaccine administration of Salmonella-killed vaccines. The growing feather (GF) cutaneous [...] Read more.
Vaccination strategies are used to manage Salmonella in chickens. Salmonella-killed vaccines are considered safer since they are inactivated. However, little is known regarding the cellular immune activities at the site of vaccine administration of Salmonella-killed vaccines. The growing feather (GF) cutaneous test has been shown to be an effective bioassay to monitor local tissue/cellular responses. We assessed local and systemic antibody responses initiated by intradermal injection of Salmonella-killed vaccines into GF-pulps of 14–15-week-old pullets. Treatments consisted of two autogenous Salmonella-killed vaccines (SV1 and SV2), S. Enteritidis (SE) lipopolysaccharide (SE-LPS), and the water-oil-water (WOW) emulsion vehicle. GF-pulps were collected before (0 h) and at 6, 24, 48, and 72 h post-GF-pulp injection for leukocyte population analysis, while heparinized blood samples were collected before (0 d) and at 3, 5, 7, 10, 14, 21, and 28 d after GF-pulp injections to assess plasma levels (a.u.) of SE-specific IgM, avian IgY (IgG), and IgA antibodies using an ELISA. Injection of GF-pulps with SV1, SV2, or SE-LPS, all in a WOW vehicle, initiated inflammatory responses characterized by the recruitment of heterophils, monocytes/macrophages, and a few lymphocytes. The WOW vehicle emulsion alone recruited more lymphocytes than vaccines or SE-LPS. The SV1 and SV2 vaccines stimulated Salmonella-specific IgM and IgA early, while IgG levels were greatly elevated later during the primary response. Overall, SV1 and SV2 stimulated a heterophil and macrophage-dominated local inflammatory- and SE-specific humoral response with an isotype switch from IgM to IgG, characteristic of a T-dependent primary antibody response. This study provides comprehensive information on innate and adaptive immune responses to autogenous Salmonella-killed vaccines and their components that will find application in the management of Salmonella in poultry. Full article
(This article belongs to the Special Issue Veterinary Vaccines and Host Immune Responses)
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<p>T cell infiltration profiles in response to injection of autogenous <span class="html-italic">Salmonella</span>-killed vaccines (SV1 and SV2), <span class="html-italic">S</span>. Enteritidis lipopolysaccharide (SE-LPS), or vehicle into the pulp of growing feathers. Twenty-four growing feathers (GF) of 14 to 15 wk old Light-brown Leghorn (LBL) pullets from (<b>A</b>) Trial 2-PHL and (<b>B</b>) Trial 3-Farm were injected with 10 µL of SV1, SV2, SE-LPS, or vehicle (water-oil-water emulsion). Injected GFs from each chicken were collected before injection (0 h) and at 6, 24, 48, and 72 h post-GF injection for leukocyte population analysis. Pulp cell suspensions were prepared from each GF, immunofluorescently stained with fluorescence-conjugated mouse monoclonal antibody (Southern Biotech) to chicken CD3 (T cells), and the percentage of CD3+ pulp cells was determined by fluorescence-based flow cytometry. Data shown are mean ± SEM. For each Trial, <span class="html-italic">n</span> = 4 pullets for SV1, SV2, and LPS, and <span class="html-italic">n</span> = 3 for the vehicle. Due to interactions involving the Trial, two-way ANOVA was conducted for each Trial. Student <span class="html-italic">t</span>-test multiple means comparisons were conducted to identify Treatment (Trt) and Time (h) differences. a–c: for each time point, treatment means without a common letter are different (<span class="html-italic">p</span> &lt; 0.05); w–z: for each treatment, means at each time-point without a common letter are different (<span class="html-italic">p</span> &lt; 0.05).</p>
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<p>B cell infiltration profiles in response to injection of autogenous <span class="html-italic">Salmonella</span>-killed vaccines (SV1 and SV2), <span class="html-italic">S.</span> Enteritidis lipopolysaccharide (SE-LPS), or vehicle into the pulp of growing feathers. Forty-eight growing feathers (GF) of 14 to 15 wk old Light-brown Leghorn (LBL) pullets from Trial 2-PHL and Trial 3-Farm were injected with 10 µL of SV1, SV2, SE-LPS, or vehicle (water-oil-water emulsion). Injected GFs from each chicken were collected before injection (0 h) and at 6, 24, 48, and 72 h post-GF injection for leukocyte population analysis. Pulp cell suspensions were prepared from each GF, immunofluorescently stained with fluorescence-conjugated mouse monoclonal antibody (Southern Biotech) to chicken Bu-1 (B cells), and the percentage of Bu-1+ pulp cells determined by fluorescence-based flow cytometry. Data shown are mean ± SEM. For each Trial, <span class="html-italic">n</span> = 8 pullets for SV1, SV2, and LPS, and <span class="html-italic">n</span> = 6 for the vehicle. Student <span class="html-italic">t</span>-test multiple means comparisons were conducted to identify Treatment (Trt) and Time (h) differences. a–c: for each time point, treatment means without a common letter are different (<span class="html-italic">p</span> &lt; 0.05); x–z: for each treatment, time means without a common letter are different (<span class="html-italic">p</span> &lt; 0.05).</p>
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<p><span class="html-italic">Salmonella</span> Enteritidis specific IgM levels in plasma after injection of autogenous <span class="html-italic">Salmonella</span>-killed vaccines (SV1 and SV2), <span class="html-italic">S.</span> Enteritidis lipopolysaccharide (SE-LPS), or vehicle into the pulp of growing feathers. SE-specific IgM levels in the plasma of 14–15 wk old Light-brown Leghorn (LBL) pullets were measured after intradermal injection of SV1, SV2, SE-LPS, or vehicle (water-oil-water emulsion) into the pulp of growing feathers. Data shown were pooled across three Trials. Heparinized blood (1.5 mL) was collected before (0 d) and at 3, 5, 7, 10, 14, 21, and 28 d post-pulp injection. Data are means ± SEM; <span class="html-italic">n</span> = 12 pullets for SV1, SV2, and SE-LPS and <span class="html-italic">n</span> = 6 for vehicle (V). Two-way repeated measures ANOVA; Student <span class="html-italic">t</span>-test multiple means comparison-test was used to determine Treatment (Trt) and Time (Day) differences. a,b: for each time point, treatment means without a common letter are different (<span class="html-italic">p</span> &lt; 0.05); w–z: for each treatment, time means without a common letter are different (<span class="html-italic">p</span> &lt; 0.05). Note: the total SV1 and SV2 immunization doses for Trial 1 were 0.140 mL/bird, and for Trial 2 &amp; 3, 0.165 mL/bird. The total SE-LPS dose was 5 µg/bird for all Trials.</p>
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<p><span class="html-italic">Salmonella</span> Enteritidis specific IgG levels in plasma after injection of autogenous <span class="html-italic">Salmonella</span>-killed vaccines (SV1 and SV2), <span class="html-italic">S</span>. Enteritidis lipopolysaccharide (SE-LPS), or vehicle into the pulp of growing feathers. SE-specific IgG levels in the plasma of 14–15 wk old Light-brown Leghorn (LBL) pullets were measured after intradermal injection of SV1, SV2, SE-LPS, or vehicle (water-oil-water emulsion) into the pulp of growing feathers. Data shown were pooled across three Trials. Heparinized blood (1.5 mL) was collected before (0 d) and at 3, 5, 7, 10, 14, 21, and 28 d post-pulp injection. Data are means ± SEM; <span class="html-italic">n</span> = 12 pullets for SV1, SV2, and SE-LPS and <span class="html-italic">n</span> = 6 for vehicle (V). Two-way repeated measures ANOVA; Student <span class="html-italic">t</span>-test multiple means comparison-test was used to determine Treatment (Trt) and Time (Day) differences. a,b: for each time point, treatment means without a common letter are different (<span class="html-italic">p</span> &lt; 0.05); w–z: for each treatment, time means without a common letter are different (<span class="html-italic">p</span> &lt; 0.05). Note: the total SV1 and SV2 immunization doses for Trial 1 were 0.140 mL/bird, and for Trial 2 &amp; 3, 0.165 mL/bird. The total SE-LPS dose was 5 µg/bird for all Trials.</p>
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<p><span class="html-italic">Salmonella</span> Enteritidis specific IgA levels in plasma after injection of autogenous <span class="html-italic">Salmonella</span>-killed vaccines (SV1 and SV2), <span class="html-italic">S.</span> Enteritidis lipopolysaccharide (SE-LPS), or vehicle into the pulp of growing feathers. SE-specific IgA levels in the plasma of 14–15 wk old Light-brown Leghorn (LBL) pullets were measured after intradermal injection of SV1, SV2, SE-LPS, or vehicle (water-oil-water emulsion) into the pulp of growing feathers. Data shown were pooled across three trials. Heparinized blood (1.5 mL) was collected before (0 d) and at 3, 5, 7, 10, 14, 21, and 28 d post-pulp injection. Data are means ± SEM; <span class="html-italic">n</span> = 12 pullets for SV1, SV2, and SE-LPS and <span class="html-italic">n</span> = 6 for vehicle (V). Two-way repeated measures ANOVA; Student <span class="html-italic">t</span>-test multiple means comparison-test was used to determine Treatment (Trt) and Time (Day) differences. a–c: for each time point, treatment means without a common letter are different (<span class="html-italic">p</span> &lt; 0.05); w–z: for each treatment, time means without a common letter are different (<span class="html-italic">p</span> &lt; 0.05). Note: the total SV1 and SV2 immunization doses for Trial 1 were 0.140 mL/bird, and for Trial 2 &amp; 3, 0.165 mL/bird. The total SE-LPS dose was 5 µg/bird for all Trials.</p>
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2 pages, 1068 KiB  
Correction
Correction: Khan et al. Oral Immunization of Chickens with Probiotic Lactobacillus crispatus Constitutively Expressing the α-β2-ε-β1 Toxoids to Induce Protective Immunity. Vaccines 2022, 10, 698
by Mohammad Zeb Khan, Fengsai Li, Xuewei Huang, Muhammad Nouman, Roshna Bibi, Xiaolong Fan, Han Zhou, Zhifu Shan, Li Wang, Yanping Jiang, Wen Cui, Xinyuan Qiao, Yijing Li, Xiaona Wang and Lijie Tang
Vaccines 2024, 12(10), 1158; https://doi.org/10.3390/vaccines12101158 - 11 Oct 2024
Viewed by 187
Abstract
The authors would like to make the following corrections to this published paper [...] Full article
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Figure 8

Figure 8
<p>Histopathological changes in immunized chickens after a challenge with the α-β2-ε-β1 fusion protein. Intestinal sections of (<b>A</b>) control group without challenge, (<b>B</b>) PBS group post-challenge, (<b>C</b>) <span class="html-italic">L. crispatus</span> N-11 group post-challenge, (<b>D</b>) pPG-T7g10-PPT/<span class="html-italic">L. crispatus</span> N-11 group post-challenge, and (<b>E</b>) pPG-E-α-β2-ε-β1/<span class="html-italic">L. crispatus</span> N-11 group post-challenge.</p>
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16 pages, 1382 KiB  
Communication
Determination of Plasmalogen Molecular Species in Hen Eggs
by Taiki Miyazawa, Ohki Higuchi, Ryosuke Sogame and Teruo Miyazawa
Molecules 2024, 29(20), 4795; https://doi.org/10.3390/molecules29204795 - 10 Oct 2024
Viewed by 294
Abstract
(1) Background: Plasmalogens are vinyl ether-type glycerophospholipids that are characteristically distributed in neural tissues and are significantly reduced in the brains of individuals with dementia compared to those in healthy subjects, suggesting a link between plasmalogen deficiency and cognitive decline. Hen eggs are [...] Read more.
(1) Background: Plasmalogens are vinyl ether-type glycerophospholipids that are characteristically distributed in neural tissues and are significantly reduced in the brains of individuals with dementia compared to those in healthy subjects, suggesting a link between plasmalogen deficiency and cognitive decline. Hen eggs are expected to be a potential source of dietary plasmalogens, but the details remain unclear. (2) Methods: We evaluated the fresh weight, dry weight, total lipid, neutral lipids, glycolipids, and phospholipids in the egg yolk and egg white of hen egg. Then, the molecular species of plasmalogens were quantified using HPLC-ESI-MS/MS. (3) Results: In egg yolk, the total plasmalogen content was 1292.1 µg/100 g fresh weight and predominantly ethanolamine plasmalogens (PE-Pls), specifically 18:0/22:6-PE-Pls, which made up 75.6 wt% of the total plasmalogen. In egg white, the plasmalogen content was 31.4 µg/100 g fresh weight and predominantly PE-Pls, specifically 18:0/20:4-PE-Pls, which made up 49.6 wt% of the total plasmalogen. (4) Conclusions: Plasmalogens were found to be more enriched in egg yolk than in egg white. It was found that humans are likely to ingest almost 0.3 mg of total plasmalogens from one hen egg. These findings highlight the importance of plasmalogens in the daily diet, and it is recommended to explore the impact of long-term dietary plasmalogen intake to assess its effect on human health. This provides a viewpoint for the development of new food products. Full article
(This article belongs to the Special Issue Progress in Molecular Spectroscopy)
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Figure 1

Figure 1
<p>Photograph of the thin-layer chromatography development of the egg yolk phospholipid fraction. In total, 30 vol% sulfuric acid aqueous solution was used as the chromogenic reagent. Egg-PL, egg yolk phospholipid fraction; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PS, phosphatidylserine.</p>
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<p>Standard curves of PE-Pls: (<b>A</b>) 18:0/18:1-PE-Pls, (<b>B</b>) 18:0/20:4-PE-Pls, (<b>C</b>) 18:0/20:5-PE-Pls, and (<b>D</b>) 18:0/22:6-PE-Pls. Standard curves were generated using Analyst<sup>®</sup> software version 1.6.3 (SCIEX, Framingham, MA, USA) within a concentration range of 10 to 1000 ng/mL. MRM for each standard were as follows: 18:0/18:1-PE-Pls (730 &gt; 339), 18:0/20:4-PE-Pls (752 &gt; 361), 18:0/20:5-PE-Pls (750 &gt; 359), and 18:0/22:6-PE-Pls (776 &gt; 385). PE-Pls, ethanolamine plasmalogens.</p>
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<p>Standard curves of PC-Pls: (<b>A</b>) 18:0/18:1-PC-Pls, (<b>B</b>) 18:0/20:4-PC-Pls, and (<b>C</b>) 18:0/22:6-PC-Pls. Standard curves were generated using Analyst<sup>®</sup> software version 1.6.3 (SCIEX, Framingham, MA, USA) within a concentration range of 10 to 1000 ng/mL. MRM for each standard were as follows: 18:0/18:1-PC-Pls (772 &gt; 184), 18:0/20:4-PC-Pls (794 &gt; 184), and 18:0/22:6-PC-Pls (818 &gt; 184). PC-Pls, choline plasmalogens.</p>
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<p>Extracted ion chromatograms from HPLC-ESI-MS/MS analysis of (<b>A</b>) analytical standard mixture (100 ng/mL), (<b>B</b>) egg yolk phospholipid fraction, and (<b>C</b>) egg white total lipid extract. MRM for each standard were as follows: 18:0/18:1-PE-Pls (730 &gt; 339), 18:0/20:4-PE-Pls (752 &gt; 361), 18:0/20:5-PE-Pls (750 &gt; 359), 18:0/22:6-PE-Pls (776 &gt; 385), 18:0/18:1-PC-Pls (772 &gt; 184), 18:0/20:4-PC-Pls (794 &gt; 184), 18:0/22:6-PC-Pls (818 &gt; 184). PC-Pls, choline plasmalogens; PE-Pls, ethanolamine plasmalogens.</p>
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<p>Amount of plasmalogens per 100 g of (<b>A</b>) fresh or (<b>B</b>) dried weight of egg yolk or egg white. Black bars represent egg yolk, and white bars represent egg white. For the comparison between egg yolk and egg white, Student’s <span class="html-italic">t</span>-test was used (* <span class="html-italic">p</span> &lt; 0.01). “Total” refers to the sum of quantifiable plasmalogens (18:0/18:1-, 18:0/20:4-, and 18:0/22:6-PE-Pls for egg yolk; 18:0/18:1-, 18:0/20:4-, and 18:0/22:6-PE-Pls and 18:0/20:4-PC-Pls for egg white). Data are expressed as mean ± standard deviation (S.D.). Experiments were conducted independently in quadruplicate. Additionally, each independent sample was analyzed in technical quadruplicate. D.W., dried weight; F.W., fresh weight; PC-Pls, choline plasmalogens; PE-Pls, ethanolamine plasmalogens.</p>
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