Volatile profile and multivariant analysis of Sanhuang chicken breast in combination with Chinese 5-spice blend and garam masala

Sanhuang chicken is a popular native breed in China and well-known for delicious flavour.Spices could enhance the chicken meat flavour and work well in preservation.Chinese 5-spice blend(CS)and garam masala(GM)are routinely using spices in China and Pakistan,respectively.The flavour profiles of Sanhuang chicken breast(CB)and its blends with CS and GM were obtained by electronic nose(E-nose),solid-phase microextraction gas chromatography-mass spectrometry(SPME GC-MS)and GC-ion mobility spectrometry(GC-IMS).Principal component analysis(PCA)efficiently discriminated the aroma profiles of three chicken formulations.The GC-chromatographs revealed the significant aroma alterations of chicken breast meat after marination with spices.Aldehydes were the major contributors of chicken aroma,while most of the aromatic hydrocarbons were generated by spices.Almost all chicken key-compounds produced by oxidation reaction were either reduced or eliminated by marination,showing the antioxidation capacity of spices leading to meat preservation.GC-IMS is not only a rapid and comprehensive detection method,but also proved to be more sensitive than GC-MS.The substantial role of both traditional spices in enhancing flavour quality of chicken meat,and their exposure as functional ingredients in Chinese and Pakistan cuisines could lead to the cross-cultural meat trade opportunities.

Gut microbiota intervention attenuates thermogenesis in broilers exposed to high temperature through modulation of the hypothalamic 5‑HT pathway

Background Broilers have a robust metabolism and high body temperature,which make them less tolerant to hightemperature(HT)environments and more susceptible to challenges from elevated temperatures.Gut microbes,functioning as symbionts within the host,possess the capacity to significantly regulate the physiological functions and environmental adaptability of the host.This study aims to investigate the effects of gut microbial intervention on the body temperature and thermogenesis of broilers at different ambient temperatures,as well as the underlying mechanism involving the"gut-brain"axis.Methods Broilers were subjected to gut microbiota interference with or without antibiotics(control or ABX)starting at 1 day of age.At 21 day of age,they were divided into 4 groups and exposed to different environments for 7 d:The control and ABX groups at room temperature(RT,24±1℃,60%relative humidity(RH),24 h/d)and the control-HT and ABX-HT groups at high temperature(HT,32±1℃,60%RH,24 h/d).Results The results demonstrated that the antibiotic-induced gut microbiota intervention increased body weight and improved feed conversion in broiler chickens(P<0.05).Under HT conditions,the microbiota intervention reduced the rectal temperature of broiler chickens(P<0.05),inhibited the expression of avUCP and thermogenesisrelated genes in breast muscle and liver(P<0.05),and thus decreased thermogenesis capacity.Furthermore,the gut microbiota intervention blunted the hypothalamic‒pituitary‒adrenal axis and hypothalamic–pituitary–thyroid axis activation induced by HT conditions.By analyzing the cecal microbiota composition of control and ABX chickens maintained under HT conditions,we found that Alistipes was enriched in control chickens.In contrast,antibioticinduced gut microbiota intervention resulted in a decrease in the relative abundance of Alistipes(P<0.05).Moreover,this difference was accompanied by increased hypothalamic 5-hydroxytryptamine(5-HT)content and TPH2 expression(P<0.05).Conclusions These findings underscore the critical role of the gut microbiota in regulating broiler thermogenesis via the gut-brain axis and suggest that the hypothalamic 5-HT pathway may be a potential mechanism by which the gut microbiota affects thermoregulation in broilers.

Protective efficacy of an H5/H7 trivalent inactivated vaccine(H5-Re13,H5-Re14, and H7-Re4 strains) in chickens, ducks, and geese against newly detected H5N1, H5N6, H5N8, and H7N9 viruses

Some H5 viruses isolated in poultry or wild birds between 2020 and 2021 were found to be antigenically different from the vaccine strains(H5-Re11 and H5-Re12) used in China. In this study, we generated three new recombinant vaccine seed viruses by using reverse genetics and used them for vaccine production. The vaccine strain H5-Re13 contains the hemagglutinin(HA) and neuraminidase(NA) genes of an H5 N6 virus that bears the clade 2.3.4.4 h HA gene, H5-Re14 contains the HA and NA genes of an H5 N8 virus that bears the clade 2.3.4.4 b HA gene, and H7-Re4 contains the HA and NA genes of H7 N9 virus detected in 2021. We evaluated the protective efficacy of the novel H5/H7 trivalent inactivated vaccine in chickens, ducks, and geese. The inactivated vaccine was immunogenic and induced substantial antibody responses in the birds tested. Three weeks after vaccination, chickens were challenged with five different viruses detected in 2020 and 2021: three viruses(an H5 N1 virus, an H5 N6 virus, and an H5 N8 virus) bearing the clade 2.3.4.4 b HA gene, an H5 N6 virus bearing the clade 2.3.4.4 h HA gene, and an H7 N9 virus. All of the control birds shed high titers of virus and died within 4 days post-challenge, whereas the vaccinated chickens were completely protected from these viruses. Similar protective efficacy against H5 viruses bearing the clade 2.3.4.4 h or 2.3.4.4 b HA gene was observed in ducks and geese. Our study indicates that the newly updated H5/H7 vaccine can provide solid protection against the H5 and H7 N9 viruses that are currently circulating in nature.

Integrative analysis of miRNA and mRNA profiles reveals that gga-miR-106-5p inhibits adipogenesis by targeting the KLF15 gene in chickens

Background:Excessive abdominal fat deposition in commercial broilers presents an obstacle to profitable meat quality,feed utilization,and reproduction.Abdominal fat deposition depends on the proliferation of preadipocytes and their maturation into adipocytes,which involves a cascade of regulatory molecules.Accumulating evidence has shown that microRNAs(miRNAs)serve as post-transcriptional regulators of adipogenic differentiation in mammals.However,the miRNA-mediated molecular mechanisms underlying abdominal fat deposition in chickens are still poorly understood.This study aimed to investigate the biological functions and regulatory mechanism of miRNAs in chicken abdominal adipogenesis.Results:We established a chicken model of abdominal adipocyte differentiation and analyzed miRNA and mRNA expression in abdominal adipocytes at different stages of differentiation(0,12,48,72,and 120 h).A total of 217 differentially expressed miRNAs(DE-miRNAs)and 3520 differentially expressed genes were identified.Target prediction of DE-miRNAs and functional enrichment analysis revealed that the differentially expressed targets were significantly enriched in lipid metabolism-related signaling pathways,including the PPAR signaling and MAPK signaling pathways.A candidate miRNA,gga-miR-106-5p,exhibited decreased expression during the proliferation and differentiation of abdominal preadipocytes and was downregulated in the abdominal adipose tissues of fat chickens compared to that of lean chickens.gga-miR-106-5p was found to inhibit the proliferation and adipogenic differentiation of chicken abdominal preadipocytes.A dual-luciferase reporter assay suggested that the KLF15 gene,which encodes a transcriptional factor,is a direct target of gga-miR-106-5p.gga-miR-106-5p suppressed the posttranscriptional activity of KLF15,which is an activator of abdominal preadipocyte proliferation and differentiation,as determined with gain-and loss-of-function experiments.Conclusions:gga-miR-106-5p functions as an inhibitor of abdominal adipogenesis by targeting the KLF15 gene in chickens.These findings not only improve our understanding of the specific functions of miRNAs in avian adipogenesis but also provide potential targets for the genetic improvement of excessive abdominal fat deposition in poultry.

AA肉鸡和如皋黄鸡海马和下丘脑GR和FKBP5的表达及其与应激敏感性的关系

旨在探究肉鸡海马和下丘脑糖皮质激素受体(glucocorticoid receptor,GR)及其负反馈因子FK-506结合蛋白5(FK-506 binding protein,FKBP5)的表达是否与应激敏感性有关。选用35日龄的AA肉鸡和如皋黄鸡,连续10 d皮下注射皮质酮(corticosterone,CORT),建立慢性应激模型。对应激后AA肉鸡和如皋黄鸡的体重、采食量、行为、血浆生化指标等进行检测,比较应激敏感性的差异。通过qPCR和Western blot检测海马和下丘脑GR和FKBP5的表达,并与血浆CORT水平进行相关分析。结果表明:1)AA肉鸡在CORT处理后体重显著降低,紧张性不动持续时间和血浆CORT水平均显著增加,但如皋黄鸡变化均不显著;AA肉鸡和如皋黄鸡血液异嗜性粒细胞/淋巴细胞比率在CORT处理后均显著上调,但AA肉鸡显著高于如皋黄鸡。2)基础状态下,如皋黄鸡海马和下丘脑GR蛋白表达量显著高于AA肉鸡;CORT处理导致AA肉鸡和如皋黄鸡海马和下丘脑GR蛋白表达显著下调,但应激后如皋黄鸡GR蛋白表达量仍显著高于AA肉鸡。3)CORT诱导AA肉鸡海马和下丘脑FKBP5 mRNA和蛋白表达均显著上调,而如皋黄鸡海马和下丘脑FKBP5的上调只发生在蛋白水平,mRNA的表达量没有变化;应激后AA肉鸡FKBP5的蛋白表达量显著高于如皋黄鸡。4)血浆CORT水平与海马和下丘脑中GR蛋白表达量呈负相关,与FKBP5蛋白表达量呈正相关;海马和下丘脑中GR与FKBP5的蛋白表达量呈负相关。以上结果表明,AA肉鸡和如皋黄鸡应激敏感性的差异与海马和下丘脑GR和FKBP5的表达及其调控有关。

miRNA sequencing analysis of healthy and atretic follicles of chickens revealed that miR-30a-5p inhibits granulosa cell death via targeting Beclin1

Background:The egg production performance of chickens is affected by many factors,including genetics,nutrition and environmental conditions.These factors all play a role in egg production by affecting the development of follicles.MicroRNAs(miRNAs)are important non-coding RNAs that regulate biological processes by targeting genes or other non-coding RNAs after transcription.In the animal reproduction process,miRNA is known to affect the development and atresia of follicles by regulating apoptosis and autophagy of granulosa cells(GCs).Results:In this study,we identified potential miRNAs in the atretic follicles of broody chickens and unatretic follicles of healthy chickens.We identified gga-miR-30a-5p in 50 differentially expressed miRNAs and found that gga-miR-30a-5p played a regulatory role in the development of chicken follicles.The function of miR-30a-5p was explored through the transfection test of miR-30a-5p inhibitor and miR-30a-5p mimics.In the study,we used qPCR,western blot and flow cytometry to detect granulosa cell apoptosis,autophagy and steroid hormone synthesis.Confocal microscopy and transmission electron microscopy are used for the observation of autophagolysosomes.The levels of estradiol(E2),progesterone(P4),malondialdehyde(MDA)and superoxide dismutase(SOD)were detected by ELISA.The results showed that miR-30a-5p showed a negative effect on autophagy and apoptosis of granulosa cells,and also contributed in steroid hormones and reactive oxygen species(ROS)production.In addition,the results obtained from the biosynthesis and dual luciferase experiments showed that Beclin1 was the target gene of miR-30a-5p.The rescue experiment conducted further confirmed that Beclin1 belongs to the miR-30a-5p regulatory pathway.Conclusions:In summary,after deep miRNA sequencing on healthy and atretic follicles,the results indicated that miR-30a-5p inhibits granulosa cell death by inhibiting Beclin1.

鸡MDA5的序列分析与结构域预测

为了解鸡黑色素瘤分化相关基因-5(Melanoma differentiation-associated protein 5,MDA5)的序列和结构域,试验使用PCR方法扩增了鸡MDA5基因并构建真核表达质粒,实现了鸡MDA5蛋白的真核表达,同时利用MegAlign等软件对鸡MDA5氨基酸序列进行分析。结果显示:鸡MDA5全长3 006 bp,编码1 001个氨基酸;鸡MDA5与人、猕猴、野猪、小鼠、绿头鸭、灰雁、鸿雁、鲫鱼、草鱼的氨基酸相似性分别为62.4%、62.9%、62.7%、64.1%、86.8%、87.3%、87.4%、48.1%、46.3%;鸡MDA5与鸟类进化关系较近,与哺乳动物次之,与鱼类进化关系较远;鸡MDA5由N端两个半胱天冬酶激活招募结构域、DExD/H box RNA解旋酶结构域和C端结构域组成;鸡MDA5二级结构包含α螺旋(50.95%)、延伸链(11.19%)、β转角(3.9%)和无规则卷曲(33.97%),三级结构与人MDA5类似,在空间上呈现半闭合环状结构。研究成功鉴定了鸡MDA5基因,分析了其基因序列并预测其结构域,为深入研究鸡MDA5结构与功能及其在鸡抗病毒天然免疫应答中的作用奠定基础。

Comparison on Immune Efficacy of Avian Influenza Virus Re-5 Strain and SS Strain in Guangxi Sanhuang Chickens

[ Objective] To compare immune efficacy of avian influenza virus （AIV） Re-5 strain （HSN1 subtype） and SS strain （ H9 subtype） in Guangxi Sanhuang chickens. [Method] A total of 180 Sanhuang chickens were selected from a breeding chicken farm in Guilin City of Guangxi and assigned into four groups （ n = 45）. At 145 days old, the chickens in the group I and II were respectively inoculated with AIV Re-5 strain and AIV SS strain, but they were kept in cages separately. The two control groups were not inoculated. Antibody levels were determined at 170 days old. [ Result] The level of antibodies against AIV Re-5 strain was significantly higher in the chickens of the group I and group II than in the corresponding control groups, respectively （ P 〈 0.01 ）. The level of antibodies against AIV SS strain was not significantly different between the group I and its control group or between the group II and its control group. [ Conclusion] The antibody level of chickens can be increased by the AIV Re-5 strain but not by the AIV SS strain.

鸡PPARγ基因转录本5的两个上游开放阅读框功能分析

前期研究发现,鸡PPARγ基因转录本5(c PPARγ5)的5’非翻译区(5’untranslated region,5’UTR)存在两个上游开放阅读框(uORF1和uORF2)。为揭示这两个uORFs在鸡PPARγ基因表达转录后调控作用,试验使用报告基因、定点突变、q RT-PCR和Western blot等技术,分析两个uORFs对报告基因活性及PPARγmRNA和蛋白表达的影响。结果表明,与野生型相比,uORF1和uORF2分别突变和同时突变均显著促进报告基因活性及PPARγm RNA和蛋白表达(P<0.05)。mRNA稳定性分析发现,两个uORFs突变对报告基因mRNA稳定性无显著影响。启动子活性分析发现,c PPARγ的5’UTR区具有启动子活性。研究结果表明,cPPARγ5的两个u ORFs抑制其翻译。

Hematomas and Limb Skeletal Malformations in Chicken Embryos Following Exposure to 5-Fluoro-2'-Deoxyuridine

Vascular injury or interruption may play a role in vertebrate limb teratogenesis. Since 5fluoro- 2'- deoxyuridine (FdU) can cause vascular injury in the murine limb and skull prior to the appearance of skeletal malformations in these structures, we studied the effects of this chemical on skeletal development in the chick embryo and noted any vascular injury. The yolk sacs of day three ehick embryos (Hamburger and Hamilton states 17-19) were injected with solutions of vary concentrations of FdU in saline. The embryos developed until the 10th day of incubation when they were fixed for study. Uninjected, saline injected, and sham injected control embryo were similarly fixed. Upon gross inspection, frequent diffuse and saccular hernatomas, as well as fluid-filled blisters, were noted in the limbs of embryos treated with FdU. After the embryos were fixed and cleared, and the skeletons stained, significant skeletal malformations were observed in these limbs. Bony elements of both the upper and lower limbs were affected in at least some of the embryos. The combination of FdU-induced hematomas and blisters with associated skeletal malformations in the same regions of some embryos suggests a relationship between these phenomena.

鸡8个功能基因5′-侧翼区与内含子的SNP多样性比较

本研究旨在探讨鸡功能基因5′-侧翼区的单核苷酸多态性(SNP)水平。作者以白来航鸡、隐性白洛克鸡、杏花鸡、丝羽乌骨鸡、固始鸡、红色原鸡为材料,扩增了GH、DDBC1、RIKEN、RASGRP3、IGF1、THRSP、VIP及PRL共8个基因的5′-侧翼区DNA序列。扩增序列全长为8,399bp,SNP的总数为161个,平均每52bp出现一个SNP。8个功能基因5′-侧翼区核苷酸多样性的θ均值和π均值分别为0.00620±0.00110和0.00559±0.00100。比较检验表明,5′-侧翼区的SNP多样性显著低于内含子区域。5′-侧翼区是基因表达的一个重要调控区域,在分子进化和系统发生过程中承受着比内含子区域更大的选择压,其较低的SNP多样性是适应性较好的表现。Tajima检验与Fu和Li检验表明,与鸡繁殖性状显著关联的VIP基因和PRL基因很可能是人工选择或自然选择的目的基因。

鸡GDF-5基因外显子1多态性与鸡骨骼发育性状的相关研究

根据GenBank发表的鸡GDF-5基因的mRNA序列设计引物,以白耳鸡和东北农业大学选育的肉鸡高、低腹脂系第8世代鸡群为试验材料,通过测序和PCR-SSCP的方法进行SNP检测和基因型分析,探讨GDF-5基因多态性与鸡生长和骨骼发育性状之间的关系。结果发现在GDF-5基因外显子1的84 bp处存在1个C/T的突变位点,对该突变位点在研究群体中进行基因型分析,结果产生3种基因型,AA型个体的基因序列和GenBank(Acces-sion No:AF123389)中的一致为C,而BB型个体的基因序列在84 bp处突变为T。基因型与鸡体组成性状的统计分析结果表明,AA基因型个体的跖骨围显著高于AB基因型个体(P<0.05);AA基因型和AB基因型个体的跖爪重和跖爪率显著高于BB基因型个体(P<0.05);AB基因型个体的股骨长和股骨重显著高于BB基因型个体(P<0.05);表明该基因对鸡的骨骼性状有较大的影响或与控制骨骼发育性状的主效基因相连锁。

鸡生长激素基因5′端部分调控区的克隆分析

利用PCR技术扩增、克隆、测序了7个跨越不同生长速度的鸡品种(系)的生长激素(GH)基因的5′端部分调控区。这7个品种(系)分别为:高生长速度的宝罗肉鸡父本;较高生长速度和一定的产蛋性能的宝罗肉鸡母本;肉蛋兼用型的芦花鸡;中等生长速度和中等体重的蛋鸡品种洛岛红和农大褐;生长速度较慢体重较轻的蛋鸡品种北京白鸡和生长速度很慢但又不是矮小型的地方品种丝毛乌骨鸡。所扩增的片段长度为760bp,包括了转录起始位点5′端侧翼区的473bp、两个可能的TATA框、组织特异性转录因子结合位点、第一个外显子和部分第一内含子。所有克隆的鸡GH基因5′端调控区与Tanaka发表的序列相比,均在-34碱基的位置上缺失一个胞嘧啶C,在+160与+161碱基之间多1个胸腺嘧啶丁,在+175碱基的位置上出现了以腺嘌呤A替换了鸟嘌呤G的情况。7个品种(系)之间的比较显示,鸡GH基因启动区具有极高的保守性,在具有不同生长速度的鸡品种间不存在任何差异。说明鸡的不同生长速度和成年体重,不是由于生长激素5′调控区变异造成的。鸡生长激素基因表达的差异可能受到内含子或3′端侧翼序列的影响。

鸡卵清蛋白基因5′端调控区的克隆及其序列分析

应用PCR技术从鸡输卵管组织基因组中扩增出了 1.0 0kb的鸡卵清蛋白基因 5′端调控区序列。序列分析表明 ,该区域含有TATA框及激素识别位点 ,具备输卵管定位表达的调控能力。为外源基因在鸡输卵管中的定位表达奠定了基础。

5-羟色胺对鸡新城疫疫苗抗体效价的影响

研究了5-羟色胺(5-HT)对鸡新城疫疫苗抗体效价的影响,结果表明:5-HT受体激动剂中剂量组的抗体效价明显高于对照组,且呈现一直上升的趋势;5-HT受体拮抗剂中剂量组的抗体效价明显低于对照组,且呈现一直下降的趋势;这表明5-HT对新城疫疫苗的抗体效价有明显的调节作用。

鸡卵清溶菌酶5′端激素受体结合位点的克隆与序列分析

应用PCR技术从鸡输卵管组织基因组中扩增出0.35kb的鸡卵清溶菌酶5′端激素受体结合位点的基因片段。序列分析表明,该区域含有2个睾酮激素受体识别位点和2个皮质酮激素受体识别位点,具备输卵管定位表达调控增强能力,为构建融合定位表达启动子调控外源基因在鸡输卵管定位表达奠定了基础。

鸡α-珠蛋白基因5′端MAR调控GFP基因在COS7细胞中表达的研究

目的研究鸡α-珠蛋白基因5′端核基质结合区(matrix association region,MAR)的基因表达调控作用。方法采用PCR方法扩增鸡α-珠蛋白基因5′端1 600 bp长度的MAR,并将该MAR连接在pCMV/GFP载体CMV启动子上游和GFP报告基因下游,构建含2个MAR的真核表达载体pGFP/2MAR。采用Lipofectimine脂质体介导法转染COS7细胞,用荧光显微镜和流式细胞术对荧光表达进行观察、定量。结果荧光显微镜观察pGFP/2MAR在转染48 h后荧光表达量明显高于pCMV/GFP的荧光表达量;流式细胞仪分析表明,在COS7细胞中pCMV/GFP和pGFP/2MAR的荧光表达量分别为17.9%和33.6%。结论该MAR确实能提高GFP在真核细胞中的表达量。

胃肠炎肉鸡肠黏膜及血清抗氧化因子和5-羟色胺变化规律研究

为阐明胃肠炎肉鸡的肠黏膜和血清抗氧化指标SOD、GSH-Px、T-AOC和MAD及炎症因子5-羟色胺(5-HT)的含量变化规律。本实验以自然发病的胃肠炎肉鸡为研究对象,采用ELISA法测定十二指肠、空肠、回肠和血清中的SOD、GSH-Px、T-AOC和MAD以及5-HT的含量。实验结果显示,胃肠炎肉鸡的消化道和血清中的SOD、GSH-Px和T-AOC在数值上都低于健康对照组,而MAD的含量高于健康对照组,绝大部分指标差异达到显著水平(P<0.05),其中十二指肠的抗氧化功能最低。胃肠炎肉鸡血清5-HT含量显著高于健康肉鸡(P<0.05);A组空肠和回肠,B、C组十二指肠和空肠5-HT含量显著高于健康对照组(P<0.05);说明胃肠炎肉鸡的血清和肠黏膜5-HT含量明显上升,抗氧化功能显著下降,活性氧代谢产物明显增加。

鸡β-防御素5在大肠杆菌中的串联表达及生物学活性检测

为提高蛋白表达量,研究选用串联表达技术对鸡β-防御素5(Av BD-5,也称Gal-5)重组蛋白进行表达及其生物学活性检测。按照NCBI上鸡β-防御素5的c DNA编码区序列,优化其基因序列后串联得到2×Gal-5,将2×Gal-5与p ET-28a双酶切后连接为重组表达载体p ET-28a-2×Gal-5,之后将p ET-28a-2×Gal-5转化到大肠杆菌(BL21)中诱导表达蛋白;重组蛋白表达成功后对其进行纯化以及体外抗菌活性的测定。结果表明,重组蛋白2×Gal-5在BL21中成功表达,分子质量约为12 ku,主要以包涵体形式存在;纯化后的重组蛋白对大肠杆菌有明显的抗菌活性,对沙门氏菌、金黄色葡萄球菌和链球菌抗菌活性较弱。试验成功构建可串联表达Gal-5且具有生物学活性的大肠杆菌原核表达载体。

硒缺乏对鸡血清组胺和5-羟色胺含量的影响

为探讨硒缺乏对鸡血清中组胺和5-羟色胺浓度变化的影响,将40只鸡随机分为低硒试验组(饲喂低硒日粮0.032mg/kg)和正常对照组(硒含量为0.229mg/kg)。分别在30、45、60和75日龄断头处死进行取样,用ELISA法对雏鸡血清中组胺和5-羟色胺浓度进行检测和对比分析。结果表明:缺硒组血清中组织胺和5-羟色胺浓度与对照组差异极显著(P<0.01),缺硒组不同日龄间差异极显著(P<0.01)。在整个试验期内,缺硒组血清中硒含量和组胺浓度呈时间-效应关系;随日龄增加,缺硒组5-羟色胺浓度逐渐升高,在60日龄时达高峰而后降低,而对照组在60日龄后则趋于平稳。缺硒刺激肥大细胞脱颗粒,释放炎性介质组胺,使血清中浓度升高。但5-羟色胺属中枢神经递质,严重缺硒引起神经损伤,使其分泌受抑,含量降低。