Comparison of heat production and plasma lipid metabolites between meat- and egg-types of Nagoya breed chicken during embryonic development

We compared heat production (HP) and plasma lipid metabolites between meat‐ and egg‐types of Nagoya breed chickens during embryonic development. To investigate HP and respiratory quotient, oxygen consumption and carbon dioxide production were measured using an open‐circuit calorimeter system. HP was significantly lower in meat‐ than in egg‐type chickens during embryonic development, and HP gradually decreased with developmental stage in both types. The respiratory quotient was constant at approximately 0.68 at every embryonic stage investigated, and the value was similar in both types. Plasma concentrations of triacylglycerol, nonesterified fatty acid, glycerol and D‐3‐hydroxybutyrate, which are associated with lipid metabolism, were similar in both types during embryonic development. These results demonstrate that chicken embryos selected for rapid growth of Nagoya breed have characteristic lower HP, and that when selecting chickens for rapid growth, HP is an important parameter during embryonic stages.     

Hepatic lipolysis in broiler chickens with different fat deposition during embryonic development

The aim of this study was to explore the hepatic lipolysis in broiler chickens with different fat deposition during embryonic development. The mRNA expression of CPT-1 (carmitine palmtoyltransferase-1), PPARα (peroxisome proliferator-activated receptor alpha) and LPL (lipoprotein lipase) genes were determined using Real time RT-PCR. The start of incubation was called day 1 (E1) and after hatching called day 1 (H1). On incubation days 9 (E9), 14 (E14) and 19 (E19) as well as at hatching (H1), samples of liver were collected. Blood samples were obtained during days 14 (E14) and 19 (E19) of embryonic development and at hatching. This study showed that serum TG (triglycerol) decreased and TC (total cholesterol) and NEFA (non-estered fatty acid) increased during embryonic development. The expression of CPT-1, PPARα and LPL genes exhibited different developmental changes. For example, little LPL gene was expressed at hatching and PPARα gene expression peaked before hatching. However, CPT-1 gene exhibited no significance during the embryonic development. Our results showed that expression of these genes in Arbor Acres (AA) broilers was significantly higher than that in San Huang (SH) broilers. Therefore, this study suggested that hepatic lipolysis in broiler chickens exhibited developmental changes during embryogenesis and breed difference which may be one of the factors in the fat deposition difference between fat line and lean line broilers during embryonic development.     

Changes in collagen fiber content and hepatic stellate cell distribution in the liver of chick embryos and growing chickens

The content of collagen and the distribution of hepatic stellate cells (HSCs) were studied to elucidate the occurrence of sex‐dependent variations in the liver of developing embryos and growing chickens. Chick embryos from embryonic days (e) 12 to e20 and chicks at 1, 4 and 8 weeks were analyzed. Liver tissue was processed using NaOH maceration and freeze‐dried to obtain the collagen fiber specimens. HSCs were identified by double fluorescent immunohistochemistry for desmin and vimentin. There were no sex‐dependent variations in the percentage of collagen fiber per liver weight and HSC area during embryonic stages. However, the content of collagen fiber increased during embryonic development in both sexes. On the other hand, the area of HSCs significantly increased in growing males but did not show any change in females. Importantly, sex differences were observed in both collagen fiber content and HSC area in the liver at 8 weeks. These results indicate that the occurrence of collagen content variations takes place at 8 weeks in chicken liver, suggesting that a sex‐dependent hormone may play an important role on the collagen production of HSCs in the growing chicken liver.     

鸡白细胞介素18双抗体夹心ELISA检测方法的建立及初步应用

应用识别不同表位的鸡白细胞介素18成熟蛋白（Mature chicken interleukin-18,mChIL-18）的2株单克隆抗体（mAb）1G9和2E6,建立检测mChlL-18的双抗体夹心ELISA,并利用此方法对禽网状内皮组织增生症病毒（Reticuloendotheliosis virus,REV）人工感染SPF鸡体内mChIL-18的分泌水平进行检测。结果显示,捕获抗体的最佳质量浓度为8mg／L,检测抗体的工作效价为1：800,待检样品的最佳稀释度为1：400,检测敏感度可达31．5ng／L,与其他细胞因子等抗原蛋白无交叉反应;跟对照组相比,REV感染鸡体内mChIL-18的表达量在7、14、21、28、35、42、49d均呈现升高,但只有14日龄时表现差异显著（P〈0．05）。结果表明,本试验成功建立了ChIL-18的双抗体夹心ELISA,为鸡传染病的细胞免疫学研究提供了可靠方法。     

胚胎及新生期蛋鸡和肉鸡垂体生长激素基因表达的比较研究

本文采用Northern杂交分析法研究了14胚龄、18胚龄、1日龄、5日龄及10日龄肉鸡和蛋鸡垂体生长激素的基因表达，同时采用放射免疫分析法（RIA）测定了垂体内生长激素的含量。结果表明：14胚龄时在肉鸡和蛋鸡垂体总RNA中均未出生长激素mRNA(GH mRNA)。从18胚龄开始可检测出一条0.8kb的GHmRNA，并且垂体GHmRNA水平的发育性变化在品种间呈现不同的规律：蛋鸡从18胚龄到10日龄垂体GHmRNA水平不断升高，日龄间差异显著（P＜0.05）。肉鸡从18胚龄到出壳1日龄，GHmRNA水平有较大幅度的升高，但从1日龄至10日龄维持在1日龄时的水平。18胚龄肉鸡垂体GH mRNA水平显著高于蛋鸡（P＜0.05），并在1日龄和5日龄均维持在较高水平，与生长速度呈正相关；而10日龄时垂体HGmRNA水平的品种差异发生逆转，蛋鸡GHmRNA水平反而高于肉鸡。垂体HG含量的发育性变化趋势与垂体HGmRNA水平相一致，10日龄时蛋鸡垂体GH含量显著高于肉鸡（P＜0.05），与生长速度呈相反的趋。     

藏鸡胚死亡曲线分析研究

本文通过对四批藏鸡胚死亡曲线进行统计,分析出：引起藏鸡胚死亡的原因除种鸡的健康状况、种蛋品质、种蛋保存外,主要是高海拔低压缺氧环境和后期相对湿度较低造成的。从而为提高藏鸡种蛋孵化率提供理论依据。     

Lethality and bone alterations in chicken embryos and newly hatched chickens given bone-active agents

Studies were undertaken to assess the chicken embryo and newly hatched chicken as models for studying the effects of bone-active agents. Initially, 1,25-dihydroxycholecaliferol (1,25[OH]2D3), sodium fluoride (NaF), parathyroid extract, epidermal growth factor, and prostaglandin E2, were tested for lethality over a broad dose range. One or 3 injections of 1,25(OH)2D3 into the yolk sac of chicken embryos resulted in death of embryos given greater than or equal to 0.1 ng/injection, whereas 0.01 ng was tolerated by the embryos. Administering 1,25(OH)2D3 intraperitoneally to newly hatched chickens as a single injection or weekly for 3 weeks resulted in no deaths at doses up to 50 ng. One or 3 IV injections of 800 micrograms of NaF were lethal to embryos, whereas injections of less than or equal to 400 micrograms were tolerated by the embryo. Giving chickens feed and water containing 2.4 g of NaF/kg was lethal, but no deaths occurred when chickens were given feed containing less than or equal to 1.2 g of NaF/kg. Mortality associated with the administration of epidermal growth factor to embryos was inconsistent, in that death occurred in embryos given a single injection of greater than or equal to 250 ng, but no deaths occurred in embryos given 3 injections at similar doses. Parathyroid extract and prostaglandin E2 were not lethal when administered to embryos and chickens in a single-injection or multiple-injection regimen. Overall, lethality in chicken embryos given a particular agent reflected the dose of bone-active agent injected, rather than the number of injections. Three of the bone-active agents were selected to characterize their microscopic bone effects in chicken embryos and chickens.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterisation of hepatic microsomal glucose-6-phosphatase from broiler chickens

1. The hepatic glucose-6-phosphatase (G-6-Pase) kinetic variables from chickens were studied in intact and disrupted microsomes using two substrates: glucose-6-phosphate (G-6-P) and pyrophosphate (PPi). They were studied from embryonic life to 51 d of age. 2. The phosphohydrolase activity studied in the broiler chicken liver microsomes corresponds to a true glucose-6-phosphatase. 3. The enzyme VMAX with both substrates in intact and disrupted microsomes showed 2 maxima: one in 19-d-old embryos and the other in 9-d-old chickens. Pyrophosphatase (PPase) VMAX in intact microsomes was higher than that of the G-6-Pase at all ages studied, except in 12 d embryos and 3-d-old chicks. In disrupted microsomes the VMAX of both enzymatic activities were similar. The G-6-Pase latency was high in the 19-d-old embryos and 51-d-old chickens. 4. The KM for PPi and G-6-Pase decreased when microsomes were disrupted. In intact microsomes the G-6-P KM was low in embryos and 3-d-old chicks and later increased. On the other hand, the PPi KM in intact microsomes showed little change during the animal's life and was lower than that of G-6-P. In disrupted microsomes the KM for both substrates were similar. 5. These results suggest a sequential incorporation of the G-6-Pase system components in the endoplasmic reticulum.     

Temporal expression of transforming growth factor-beta2 and myostatin mRNA during embryonic myogenesis in Indian broilers

TGF-beta2 and myostatin, the members of TGF family, act through both autocrine and paracrine mechanisms to regulate the growth and differentiation at various developmental stages in chicken. The kinetics and expression profile of these two growth factors were investigated by semi-quantitative RT-PCR, during the myogenesis of Indian broiler chickens. Total RNA was isolated from whole embryos on each of embryonic days (E) 0-6 (n=3 per day) and from the biceps femoris muscle at E7-E18 (n=3 per day). The expression of TGF-beta2 was noticed on E2 that remained at the same level until E6. In biceps femoris muscle, higher level of TGF-beta2 expression was observed during E7-E12, which decreased gradually thereafter. These findings suggested that TGF-beta2 might be a regulatory factor participating in the myogenesis of chicken embryos. Initial myostatin expression was noticed on E1, even before the myogenic lineage is established in embryo. This finding suggested an additional role of myostatin in early chicken embryo development, other than myogenesis. Furthermore, myostatin expression was significantly higher on E3 as compared to earlier studies, where initial higher level was observed at E2, suggesting the differential expression of myostatin among breeds. Higher and almost static myostatin expression was noticed in biceps femoris muscle during the entire period of myogenesis (E7-E18). In the present study, the ontogeny of myostatin expression coincided with myogenesis of chicken. Therefore, it may be hypothesized that myostatin is not only a major determinant of muscle mass, but also involved in early embryogenesis in chickens.     

interactions between escherichia coli and Newcastle disease virus in chickens

We investigated the interaction between Newcastle disease virus (NDV) and Escherichia coli in cell cultures, embryonated eggs, and 8-wk-old chickens. We measured the interactions on the basis of bacterial adherence and NDV hemagglutination titer in chickens, chicken embryos, and chicken embryo cell culture. Depending on the inoculation order of E. coli, a significant alteration of the growth of NDV was observed in both chickens and chicken embryos. When certain strains of E. coli were given before NDV exposure, the virus titers were lowered. In chickens, the mean virus titer was significantly (P < 0.05) lowered in the crop, the proventriculus, the gizzard, and the jejunum. However, there were no significant differences (P < 0.05) between the two groups for NDV titers in the duodenum, ileum, and cecum. In chicken embryos, when E. coli serotypes O78 and O119:B14 were inoculated before NDV exposure, the mean NDV titers were significantly (P < 0.5) lowered. However, there were no significant differences (P < 0.05) in NDV titer between the two groups when E. coli serotypes O78:K80:NM and O1ab:K NM were inoculated 24 hr before NDV exposure. When NDV was given prior to E. coli exposure, NDV titer was higher in both chickens and chicken embryos. In chickens, when NDV was given 48 hr before E. coli inoculation, NDV was detected in the proventriculus, gizzard, jejunum, ileum, and cecum, whereas no virus was detected in the control groups (NDV only). In the crop, NDV was detected at a significantly (P < 0.05) higher titer in the E. coli-inoculated group when compared with the control group that received NDV alone. In chicken embryos, virus titer was significantly (P < 0.05) higher when NDV was given 24 hr before E. coli inoculation for all three NDV strains used (Ulster and V4 strains). Adherence of E. coli to chicken embryo kidney (CEK) cells was significantly higher (P < 0.05) when the CEK cells were infected first with NDV and then by E. coli. The mean bacterial count per microscopic field in NDV-uninfected monolayers was eight compared with 112 for the NDV-infected monolayers. In approximately 10% of the fields in NDV-infected monolayers, the bacteria were too numerous to count.     

IBDV B87株不同鸡胚代次毒力和细胞嗜性差异的比较研究

对IBDV疫苗株:B87的鸡胚传代毒E2和E6的VP2片段进行测序和分析,发现两个代次毒株VP2序列存在7个氨基酸的差异,即S76G、L217S、Q253H、D279N、A284T、I294.L、S330R。将E2和E6接种CEF细胞,E2不能在CEF'上增殖,而接种E6的CEF细胞可以产生明显的CPE,说明两个毒株对CEF的嗜性不同。分别将E2和E6接种3周龄SPF鸡,对鸡的毒力进行比较,接种后第7天开始,E2出现囊体比下降,BB指数明显低于E6,相应的法氏囊切片经HE染色后,接种E2的法氏囊滤泡萎缩严重,结构松散,E6只有部分滤泡出现萎缩,滤泡间隔基本正常,说明E2,E6对SPF'鸡法氏囊毒力存在明显差异。根据两个毒株的VP2序列差异以及相关研究,推测Q253H/D279N/A284T三个位点突变可能是IBDV毒力和细胞嗜性差异的分子基础,但另外4个位点也可能对IBDV毒力差异产生影响。     

Expression of chicken LEAP-2 in the reproductive organs and embryos and in response to <Emphasis Type="Italic">Salmonella enterica</Emphasis> infection

In recent years host antimicrobial peptides and proteins have been recognised as key mediators of the innate immune response in many vertebrate species, providing the first line of defense against potential pathogens. In chickens a number of cationic antimicrobial peptides have been recently identified. However, although these peptides have been studied extensively in the avian gastrointestinal tract, little is known about their function in the chicken reproductive organs and embryos. Chicken Liver Expressed Antimicrobial Peptide-2 (cLEAP-2) has been previously reported to function in protecting birds against microbial attack. The aim of this study was to investigate the expression of cLEAP-2 gene in the chicken reproductive organs, as well as in chicken embryos during embryonic development, and to determine whether cLEAP-2 expression in the chicken reproductive organs was constitutive or induced as a response to Salmonella enteritidis infection. RNA was extracted from ovary, oviduct, testis and epididymis of sexually mature healthy and Salmonella infected birds, as well as from chicken embryos until day ten of embryonic development. Expression analysis data revealed that cLEAP-2 was expressed in the chicken ovary, testis and epididymis as well as in embryos during early embryonic development. Quantitative real-time PCR analysis revealed that cLEAP-2 expression was constitutive in the chicken epididymis, but was significantly up regulated in the chicken gonads, following Salmonella infection. In addition, expression of cLEAP-2 during chicken embryogenesis appeared to be developmentally regulated. These data provide evidence to suggest a key role of cLEAP-2 in the protection of the chicken reproductive organs and the developing embryos from Salmonella colonization.     

Total lipid and triacylglycerol contents in the liver of broiler and layer chickens at embryonic stages and hatching

To compare the hepatic function of broiler and layer chickens (Gallus gallus domesticus) at various embryonic stages and hatching, the total lipid and triacylglycerol (TG) content were determined. The chicken embryos accumulated a large amount of lipids in the liver and the total lipid content gradually increased toward hatching, though no significant difference was observed between broilers and layers. The TG contents in the liver increased considerably with developmental stage. At embryonic day 14 the TG content in the liver was similar between broilers and layers; thereafter, it was 1.3 and 2.2 times higher in broilers than in layers at embryonic day 18 and for newly hatched chicks, respectively. Chick embryos accumulate an excessive amount of cholesterol ester in the liver, but cholesterol ester is replaced by TG after hatching. The results of hepatic TG contents in the present study suggest that the development of the hepatic function between broilers and layers may already differ at embryonic stages.     

Detection of the EGFP sequence in breast muscle of 3‐year‐old chicken after transfection using sonoporation

In our continuing effort to generate transgenic chickens, sonoporation was chosen to insert an exogenous gene into the chicken genome. An EGFP expression vector (pCAG‐EGFPac) and microbubbles were injected into the central disc of stage‐X blastoderm or the germinal crescent of stage‐4 embryos, followed by ultrasonic vibration. Nineteen chicks out of 108 treated embryos hatched, six females and six males out of these 19 chicks grew to sexual maturity and two females and three males lived for 3 years. Genomic DNA from 17 out of 35 gonads from embryos and chicks that died before sexual maturity was EGFP‐positive by PCR. No EGFP sequence was detected in the genomic DNA of 322 embryos from six sexually mature females and the semen from four sexually mature males by PCR. When genomic DNA was obtained from various tissues of five 3‐year‐old chickens, the EGFP sequence was amplified from the genomic DNA of the breast muscle of a female (No. 85). The above sequence was subjected to DNA sequencing and verified to be the EGFP sequence. These results showed that sonoporation is an effective tool for the transduction of exogenous genes into chicken embryos for the generation of transgenic chickens.     

In ovo carbohydrate supplementation modulates growth and immunity‐related genes in broiler chickens

A study was undertaken to investigate the role of in ovo administrated carbohydrates on the expression pattern of growth and immune‐related genes. In ovo injections (n = 400) were carried out on the 14th day of incubation into the yolk sac/amnion of the broiler chicken embryos. Expression of growth‐related genes: chicken growth hormone (cGH), insulin‐like growth factor‐I & II (IGF‐I & II) and mucin were studied in hepatic and jejunum tissues of late‐term embryo and early post‐hatch chicks. Expression of candidate immune genes: Interleukin‐2, 6, 10 and 12 (IL‐2, IL‐6, IL‐10 and IL‐12), Tumour necrosis factor‐alpha (TNF‐α) and Interferon gamma (IFN‐γ) were studied in peripheral blood monocyte cells of in ovo‐injected and control birds following antigenic stimulation with sheep RBC (SRBC) or mitogen concanavalin A (Con‐A). Glucose injection significantly increased the expression of IGF‐II gene during embryonic period and both cGH and IGF‐II in early post‐hatch period, while ribose‐injected chicks had higher expression of IGF‐II gene during embryonic stage. Enhanced mucin gene expression was also observed in fructose‐injected chicks during embryonic age. Glucose‐injected chicks had higher expression of IL‐6 or IL‐10, while those injected with fructose or ribose had higher expression of IL‐2, IL‐12 and IFN gamma. It is concluded that in ovo supplementation of carbohydrates might help in improving the growth of late‐term embryos and chicks. In ovo glucose could modulate humoral‐related immunity, while fructose or ribose might help in improving the cellular immunity in broiler chickens.     

Pathogenicity of embryo-adapted serotype 2 OH strain of infectious bursal disease virus in chickens and turkeys

The pathogenicity of serotype 2 OH strain of infectious bursal disease virus (IBDV) to specific-pathogen-free (SPF) chicken embryos and 2-wk-old SPF chickens and turkey poults was investigated. The virus was pathogenic for chicken embryos after five passages as evidenced by pathologic changes in inoculated embryos. The embryo-adapted virus was not pathogenic for 2-wk-old SPF chickens and turkey poults as indicated by lack of clinical signs, gross or microscopic lesions in the bursa of Fabricius of inoculated birds. Bursa-to-body-weight ratios of the inoculated chickens and turkey poults were not significantly different from those of uninoculated controls. Virus-neutralizing antibodies to serotype 2 IBDV were detected in inoculated chickens and turkeys. Results of this study indicated that the embryo-adapted serotype 2 OH IBDV isolate that is pathogenic for chicken embryos is infectious but not pathogenic in chickens and turkeys.     

Attempt to produce nuclear transferred primordial germ cells using electrofusion in domestic chicken

As a step to develop a somatic nuclear transfer technique for avian species, an attempt to produce somatic nuclear transferred primordial germ cells (PGC) in the domestic chicken was carried out. Primordial germ cells and embryonic blood cells (EBC) were collected from 2‐day‐old embryos and the nuclei were transferred from EBC into PGC by electrofusion. The most efficient pearl chain was developed when a 350‐V/cm AC field was applied for 60 s. Cell fusion between PGC and EBC was most effective when 4‐kV/cm DC pulses, 60 µs pulse width, were applied three times to a cell suspension dispersed in 0.2 or 0.25 mol/L saccharose solution. The present results provide basic information for the production of somatic cell nuclear transferred chickens using PGC as the nuclear recipient.     

Effects of 6-hydroxydopamine on the development of the immune system in chickens

It has been suggested that the sympathetic nervous system communicates with lymphocytes expressing cell surface receptors for neurotransmitters such as norepinephrine (NE), on the basis of the finding that neurotransmitters modify immune responses in mammalian species. We confirmed that chicken lymphocytes in the brusa of Fabricius, thymus and spleen expressed beta-adrenergic receptor (beta-AR) mRNA from embryonic day (E) 10 and that intracellular cAMP level was elevated by NE, suggesting that lymphocytes express functional beta-AR on their surface at an early embryonal stage. To clarify whether the nervous system is involved in the development of the immune system, the effects of 6-hydroxydopamine (6-OHDA), one of sympathectomizing agents, on chicken lymphocytes was investigated. A single injection of 6-OHDA at a dose of 400 microg into a chicken embryo was carried out at E7 or 14 (as referred to E7 group and E14 group, respectively). NE level and the relative proportion of Bu-1a(+), CD4(+) and CD8(+) cells in the spleen of 3-week-old chickens were not altered by 6-OHDA treatment. However, the proliferative responses and expression of IL-2 mRNA in spleen cells cultured with pokeweed mitogen were reduced in E7 group compared with those of control. Furthermore, in CD8(+) spleen cells of E14 group of 3-week-old chickens, the expression of beta-AR mRNA and the relative increase of intracellular cAMP stimulated with NE were significantly decreased. These results suggest that the sympathetic nervous system affects the development of the immune system.     

接种不同剂量E.tenella对雏鸡血清抗氧化酶的影响

同一日龄雏鸡接种不同剂量的柔嫩艾美尔球虫,研究接种不同剂量柔嫩艾美尔球虫对雏鸡血清抗氧化酶的影响。将60只健康鸡随机分为6组(n=10),分别接种0、0.5×104、1×104、2×104、4×104和8×104个孢子化卵囊。接种后7 d颈静脉采血,分离血清,测定血清脂质过氧化物(MDA)含量和超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-PX)、过氧化氢酶(CAT)活性。结果表明雏鸡感染不同剂量的柔嫩艾美尔球虫后,按接种剂量从低到高,血清中MDA含量先降低后升高变化差异极显著(P0.01);血清SOD活性先升高后降低,差异极显著(P0.01);血清GSH-PX活性先升高后降低,差异极显著(P0.01);血清CAT活性较对照组极显著性降低(P0.01)。不同剂量柔嫩艾美尔球虫感染对雏鸡血清MDA含量和SOD、GSH-PX、CAT活性有一定的影响。