Estradiol inhibits hepatic stellate cell area and collagen synthesis in the chicken liver

Hepatic stellate cells (HSCs) are the main collagen‐producing cells in the liver. The HSC area and amount of collagen fibers are different between male and female chickens. This study was performed to confirm the effect of estradiol on collagen synthesis in the growing chicken liver. Blood estradiol levels in chicks were compared at 4 and 8 weeks of age, and the collagen fibril network in liver tissue was observed at 8 weeks by scanning electron microscopy. Intraperitoneal administrations of estradiol and tamoxifen to male and female chicks, respectively, were performed daily from 5 to 8 weeks of age. The areas of HSCs and collagen contents were measured in the liver tissue. The blood estradiol level was higher in females than in males, and the collagen fibril network was denser in males than in females at 8 weeks of age. Estradiol administration in males induced decreases in the HSC area and collagen content of the liver. Conversely, tamoxifen administration in females induced an increase in the HSC area but did not facilitate collagen synthesis. Based on these results, estradiol inhibits the area and collagen synthesis of HSCs in the growing chicken liver under normal physiological conditions.     

The comparison of the collagen fiber contents and hepatic stellate cell distribution in male and female chicken livers

The difference in collagen fiber content, morphological properties and hepatic stellate cell (HSC) distribution was investigated in the liver of both sexes in chicken. Collagen fiber specimens were obtained by maceration treatment with NaOH solution. HSCs were detected using desmin‐specific immunohistochemistry. The ratio of liver weight to body weight was larger in the female than the male chickens. Collagen fiber content, the numerical density of HSCs and the percentage area displaying desmin immunopositivity were not different between the right and left lobes of the liver, in both male and female chickens. However, all of these parameters were larger in the males than the females. In the light microscopic observation, many HSCs in the male had large and elongated cytoplasmic processes. Conversely, HSCs with poorly developed cytoplasmic processes were frequently observed in females. Liver tissue is structurally stronger in male chickens than females and the activity and density of HSCs may be related to the collagen fiber content in chicken liver.     

Collagen content and architecture of the pectoralis muscle in male chicks and broilers reared under various nutritional conditions

Varying chicken growth rates were induced with different nutritional regimes, and the collagen content and architecture of M. pectoralis (PT) were compared among 21‐day‐old chicks and broilers at 80 or 95 days of age. The percentage of muscle weight to live weight was higher in rapid growing chicks (8.4%) than slow growing chicks (6.3%). The 80‐day‐old broilers engaged in compensatory growth after the early slow growth period producing PT muscle at 11% of live weight. The 80‐ and 95‐day‐old chicks with restricted late growth after an early rapid growth period showed PT weight at 8% and 9% of live weight, respectively. Collagen content of the PT muscle markedly decreased from the chicks to the broilers. The collagen concentration was higher in the late‐growth restricted broilers (1.67–1.88 mg/g) than the compensatory growth broilers (1.01–1.10 mg/g). Collagen concentration did not differ between the rapid and slow growing chicks (2.72 and 2.94 mg/g). Scanning electron micrographs showed thick and thin perimysia, and honeycomb endomysia. In the perimysia, a stack layer of collagen platelets and a reticular layer of collagen fiber cords were distinguished and collagen baskets of adipocytes were observed. The perimysial collagen fibers became thicker during growth of the chicks to broilers. However, in the late‐growth restricted broilers, the perimysial collagen fibers seemed to have retarded development compared with the compensatory growth birds. The PT muscle of chickens develops optimally when body growth is enhanced. The PT muscle of the compensatory growth broilers had improved collagen architecture regardless of the marked decrease in collagen content.     

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.     

Based serum metabolomics analysis reveals simultaneous interconnecting changes during chicken embryonic development

Metabolic disorder is a major health problem and is associated with a number of metabolic diseases. Due to native hyperglycaemia and resistance to exogenous insulin, chickens as a model had used in the studies of adipose tissue biology, metabolism and obesity. But no detailed information is available about the comprehensive changes of serum metabolites at different stages of chicken embryonic development. This study employed LC/MS‐QTOF to determine the changes of major functional metabolites at incubation day 14 (E14d), 19 (E19d) and hatching day 1 (H1d), and the associated pathways of differential metabolites during chicken embryonic development were analysed using Metabolite Set Enrichment Analysis method. Results showed that 39 metabolites were significantly changed from E14d to E19d and 68 metabolites were significantly altered from E19d to H1d in chicken embryos. Protein synthesis was promoted by increasing the concentrations of L‐glutamine and threonine, and gonadal development was promoted through increasing oestrone content from E14d to E19d in chicken embryos, which indicated that serum glutamine, threonine and oestrone contents may be considered as the candidate indicators for assessment of early embryonic development. 2‐oxoglutaric acid mainly contributed to enhancing the citric cycle, and it plays an important role in improving the growth of chicken embryos at the late development; the decreasing of L‐glutamine, L‐isoleucine and L‐leucine contents from E19d to H1d in chicken embryonic development implied their possible functions as the feed additive during early posthatch period of broiler chickens to satisfy the growth. These results provided insights into understand the roles of serum metabolites at different developmental stages of chicken embryos, it also provides available information for chicken as a model to study metabolic disease or human obesity.     

Evaluation of embryonic age and the effects of different proteases on the isolation and primary culture of chicken intestinal epithelial cells in vitro

The present study evaluates the effects of embryonic age and proteolytic enzymes on the isolation and primary culture of chicken enterocyte and to establish an effective technique for chicken intestinal epithelial cell (IEC) cultivation. Fourteen‐day‐old, 16‐day‐old and 18‐day‐old embryos (average weight: 52.23 ± 0.76 g, 50.86 ± 0.99 g, 48.98 ± 1.03 g) were the source for preparation of enterocyte culture, and trypsin‐ethylene diamine tetraacetic acid, collagenase, thermolysin and combination of collagenase and thermolysin were used for digestion medium. Optimal culture protocols were determined by qualitative assays of proliferation. Cells isolated by using 14‐day‐old embryo and collagenase obtain the best attachment and growth in culture, and the production of continuously growing IEC cultures. Thus, we conclude that the use of collagenase as a dissociating enzyme and 14‐day‐old embryo as a source can be advantageously applied to the isolation of chicken IEC and this method may be useful for various applications and basic studies of the intestinal tract concerning such objects as physiology, immunology and toxicology.     

过表达FOXL2基因对鸡胚性腺分化的影响

试验旨在研究FOXL2基因对鸡胚性腺分化的影响。本试验分为试验1组、试验2组和空白对照组（鸡胚数量分别为260、100、20枚），试验组通过胚盘下腔注射的方法分别将pLV-FOXL2慢病毒重组质粒、pLV空质粒注入胚胎期第2天的鸡胚，空白对照组不做处理并与试验组一起孵化至出雏，利用CHD1基因遗传性别鉴定的方法对出雏的雏鸡进行性别检测，分析其性腺解剖学、组织学结构变化，并利用免疫组化的方法检测性腺FOXL2和CYP19A1蛋白表达量。结果显示，试验1组遗传性别为公的23只，遗传性别为母的18只，表型性别为公的21只，表型性别为母的18只，其中有2只表型性别不典型，左侧性腺发生变化，朝卵巢结构转变；试验2组遗传性别为公的9只，遗传性别为母的12只，表型性别与遗传性别一致。阳性PCR检测结果显示，试验1组获得阳性个体10个，阳性率为24.4%（10/41）；试验2组获得阳性个体8个，阳性率为38.1%（8/21）。性腺解剖学结果显示，阳性pLV-FOXL2雄性鸡胚左侧性腺体积明显大于右侧性腺，表现膨松状态；组织切片结果显示，雄性鸡胚性腺具有典型的卵巢皮质层和髓质层结构；阳性pLV-FOXL2雌性鸡胚性腺的发育无明显变化。免疫组化结果显示，FOXL2和CYP19A1蛋白在试验1组左右侧睾丸中的表达量与空白对照组母鸡卵巢中的表达量相似，显著高于试验2组（P<0.05）。以上结果表明，FOXL2基因可能促进鸡雄性性腺的性反转，在鸡性腺分化和发育过程中发挥着重要的作用。     

Effect of Overexpression of FOXL2 Gene on Gonadal Differentiation in Chicken Embryo

The aim of this study was to investigate the effect of FOXL2 gene on gonadal differentiation in chicken embryos.This test was divided into the experiment groups 1,2 and blank control group(chicken embryos were 260,100 and 20,respectively).In the experimental group,pLV-FOXL2 lentivirus recombinant plasmids and pLV empty plasmids were injected into the chicken embryo on the second day of the embryonic stage by subpaneoidal injection.The blank control group was not treated and incubated with the experimental groups until the chick was hatched.CHD1 gene genetic sex identification method was used to detect the sex of chicks,the changes of gonadal anatomy and histological structure were analyzed,and the expression levels of FOXL2 and CYP19A1 proteins were detected by immunohistochemistry.The results showed that in the experiment group 1,there were 23 male and 18 female of genetic sex,21 male and 18 female of phenotypic sex,and two of them had atypical phenotypic gender,with changes in the left gonadal gland toward ovarian structure.The phenotypic sex was consistent with the genetic sex in experiment group 2,with 9 male and 12 female.Positive PCR results showed that 10 positive individuals were obtained in the experiment group 1,with a positive rate of 24.4% (10/41),and 8 positive individuals were obtained in the experiment group 2,with a positive rate of 38.1% (8/21).The anatomical structure of the gonad showed that the volume of the left gonad was significantly larger than that of the right gonad in the positive plV-FOXL2 male embryos.The results of tissue sections showed that the gonad of male chicken embryo had typical structure of ovarian cortex and medulla.There was no significant change in the development of gonad in female embryos with positive pLV-FOXL2.Immunohistochemical results showed that the expression levels of FOXL2 and CYP19A1 proteins in the left and right testicle of experiment group 1 were similar to that in the hen ovary of the blank control group,and were significantly higher than that in experiment group 2 (P<0.05).These results suggested that FOXL2 gene might promote the sexual inversion of chicken gonads and played an important role in the differentiation and development of chicken gonads.     

Nondestructive sex-specific monitoring of early embryonic development rate in white layer chicken eggs using visible light transmission

ABSTRACT

1. Sex-specific variations in early embryonic development rates may pre-empt later variations in embryonic development through to pipping and hatching. Given that erythropoiesis (blood production) can be equated with early embryonic growth rate, it was hypothesised that blood pigment haemoglobin can act as a specific spectral fingerprint for changes in growth rate. Moreover, by measuring longitudinal, rather than lateral, spectral transmission through the egg, a more consistent spectrum with a higher signal-to-noise ratio could be captured.

2. Longitudinal visible transmission (T575/T598 ratio), which is sensitive to haemoglobin, was used to monitor sex-specific early embryonic development rate in white layer chicken eggs from d 0 to 8 of incubation. The sex of these eggs was subsequently confirmed two days after hatching.

3. Embryonic development was detectable from d 3 (72 h) of incubation, 36 h earlier than previously reported lateral spectral measurements, supporting the greater sensitivity of longitudinal measurements.

4. At d 3, the mean T575/T598 ratio for male embryos was significantly lower (P < 0.001) (i.e. higher absorbance of haemoglobin) than for female embryos, which was thought to be due to sex-differences in early embryogenesis. On the other hand, female embryos had a significantly lower (P < 0.05) mean T575/T598 ratio than male embryos at d 7 of incubation, presumably due to the combined effects of oestrogen synthesis receptors and enzymes on erythropoiesis in female embryos at this time.

5. In conclusion, the proposed methodology has the sensitivity to differentiate sex-specific embryonic development rates during early incubation and the potentiality to advance precision incubation management and poultry research.     

Developmental changes of protein profiles in the embryonic Sanhuang chicken liver

Understanding embryonic liver development bears the potential to provide important insights into treatments and preventative strategies for paediatric liver disease. Using Sanhuang (SH) chicken as a model system, we sought to identify the proteomic changes associated with embryonic liver development using differential display of proteins with two-dimensional (2-D) polyacrylamide gel electrophoresis and matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry analysis. Embryonic livers from 200 SH chicken embryos were isolated on days 9, 14 and 19 during incubation and also immediately after hatching. Six hundred and two protein spots were displayed on 2-D gels stained with colloidal Coomassie brilliant blue, of which, 25 protein spots were found to have changes up to threefold in abundance during development. We identified these spots using MALDI-TOF mass spectrometry and found 23 of 25 proteins to be associated with carbohydrate metabolism, cell division, lipid metabolism and signal transduction. Our results provide insight into the biochemical events taking place during the development of SH chicken embryonic liver and highlight the value of proteomics in characterizing complex biochemical processes. Furthermore, the proteome maps may facilitate future studies addressing the effects of genetic and environmental factors and related studies on the development and quality of chicken embryonic liver.     

Comparative observations on the growth changes of the histochemical property and collagen architecture of the Musculus pectoralis from Silky, layer-type and meat-type cockerels

Growth‐related changes in the histochemical properties and collagen architecture of the Musculus pectoralis were compared among Silky, layer‐type and meat‐type cockerels. Histochemical and immunohistochemical methods were used and collagen architecture was studied using scanning electron microscopy. The total amount of collagen present was also measured. The diameter of type IIB myofibers was similar or rather larger in the layer‐type birds compared with the meat‐type. The collagen content was generally low for 5–10 weeks across the breeds and then increased in the other breeds except for Silky. In the perimysium, the collagen bundles gradually increased in size and the density of the fibrils also increased during growth. At 30 weeks of age, the layer‐type birds showed compact collagen bundles while the meat‐type had loose bundles. The endomysial collagen network appeared relatively denser in the meat‐type chicks compared to the others at week 1. At 30 weeks of age, compact and felt‐like structure of endomysium was shown by Silky and layer‐type chickens, while the meat‐type showed a relatively loose arrangement of tissue in the endomysial collagen. From these results, it appears that the meat‐type chicken can produce a large M. pectoralis with many, relatively thinner myofibers and a relatively undeveloped form of intramuscular collagen structure.     

庄河大骨鸡育成期生长特点研究

为了解庄河大骨鸡现有群体育成期的生长特点,本研究对庄河大骨鸡育成期720余只母鸡和100余只公鸡的体重进行了3个时间点的测定,深入分析了群体的增重和变异情况。研究发现,除8w母鸡群体外,其他时间点的公母鸡群体的体重和增重变异度均超过10%,母鸡的变异度高于公雏;不仅公母雏间生长发育规律不同,而且同性别群体内部生长发育规律也呈现明显的多样性。     

Relationship between the concentration of myoglobin and parvalbumin in various types of muscle tissues from chickens

The concentration of parvalbumin was determined in various types of chicken muscle by immunological analysis and was compared with that of myoglobin. Parvalbumin was present specifically in skeletal muscle and absent in cardiac and gizzard muscle; exceptionally, neither parvalbumin nor myoglobin was detected in white breast muscle. The wing and leg red muscles, which had larger amounts of myoglobin, contained smaller quantities of parvalbumin. In these muscles, the concentration of parvalbumin was inversely related to that of myoglobin (correlation coefficient = -0.69). Both myoglobin and parvalbumin were observed in the legs of 18-d-old embryos; the parvalbumin content exceeded that of myoglobin until the birds were 4 to 6 weeks old, but the relationship was reversed thereafter. Myoglobin in gizzard muscle was present in 18-d-old embryos and increased markedly at hatching; it was already present in cardiac muscle at an early embryonic stage, increasing gradually until 14 weeks after hatching.     

Contribution of insulin to the ascorbate recycling system in the chicken liver

The effect of insulin on the ascorbate recycling system in the chicken liver was examined. First, insulin was injected subcutaneously into the chicken, and liver glutathione‐dependent dehydroascorbate reductase (GSH‐DHAR) activity was determined. Insulin increased liver GSH‐DHAR activity, but did not affect plasma and liver ascorbate concentration. Dehydroascorbate increased plasma and liver ascorbate levels, and liver GSH‐DHAR activity. However, distinct changes in plasma insulin level were not observed by dehydroascorbate injection. In addition, reduction of external dehydroascorbate in cultured chicken hepatocytes could not be observed in an insulin‐deprived culture, although the cells reduced external dehydroascorbate in a serum‐free culture with insulin. We concluded that insulin affects the ascorbate recycling system as an essential factor in the chicken liver.     

Increased collagen III in culled chicken meat after feeding dietary wood charcoal and vinegar contributes to palatability and tenderness

We comprehensively evaluated meat quality in chickens fed a diet consisting of wood charcoal and vinegar (WCV) using food scientific and histological approaches. In culled hens, lipid and fatty acid in Musculus semimembranosus, cooking loss and sensory tests of whole thigh meat, and meat texture of breast meat were observed. In male broilers, cross section of M. semimembranosus was used for observations on muscle area, perimysium, non‐collagen total protein and total collagen content, and anti‐collagen I and III reactions. In frozen male broilers, conventional morphology of M. semimembranosus as well as chicken anti‐collagen III reaction to selected muscles of thigh meat and breast meat were compared between the control and WCV‐fed birds. Increased lipid and fatty acids, decreased cooking loss, high score in total evaluation for sensory test of thigh meat, and decreased meat texture values were observed for culled hens fed WCV. The higher values of muscle area, total collagen and collagen III were observed for broilers fed WCV. No perimysium collapse for M. semitendinosus or increased collagen III reactions of M. tensor fasciae latae, the flexor muscle group and M. pectoralis superficialis were observed for frozen muscles in the WCV group. These total results suggest that WCV produces palatable and tender meat by increasing collagen III.     

早期鸡胚发育过程中增殖细胞核抗原在法氏囊的表达

法氏囊是鸟类特有的器官,是B淋巴细胞成熟的场所.本研究分析了鸡胚早期发育过程的中法氏囊形态变化及增殖细胞核抗原PCNA表达变化.结果表明鸡胚6.5 d(E6.5)时,法氏囊开始出现在鸡胚泄殖腔的最深处,从最开始E6.5的一些小的囊泡发育成E8的一个大腔,E8～E10,法氏囊继续发育.从E11开始,法氏囊开始出现一些小的皱褶,E11～E18,这些小的皱褶逐渐变得清晰,并贯穿整个法氏囊.在E9时,法氏囊轮廓形成,可以发现在表皮层和基膜层PCNA有着广泛的表达.统计分析表明表皮层的PCNA阳性率在100％左右,在整个发育早期几乎没有变化；而基层的阳性率随着胚胎的发育逐渐下降,在E9、E10、E11、E12、E14和E18表达率分别是97％、93％、71％、55％、56％和20％.     

鸡肉嫩度评定方法及其指标间的相关分析

采用剪切值、胶原蛋白(总胶原蛋白、热溶性胶原蛋白、热残留胶原蛋白)和肌纤维结构(肌纤维密度、肌纤维直径)3种方法,分别评定了7个不同鸡种的嫩度。结果表明,应用3种评定方法可以划分出鸡种间的嫩度等级,但各指标间的嫩度排序有差异。相关分析表明,剪切值、总胶原蛋白、热残留胶原蛋白和肌纤维直径是嫩度评定的主要指标。其中剪切值与热残留胶原蛋白、热残留胶原蛋白与肌纤维直径和总胶原蛋白与热残留胶原蛋白为极显著正相关(P<0 01),剪切值与肌纤维直径为显著正相关(P<0 05)。经综合分析,剪切值的分辨率最高,热残留胶原蛋白、肌纤维直径综合代表性较强。以上评定结果与感官评定的嫩度对比有一定差异,感官评定与组织结构评定的结果相近,与剪切值、胶原蛋白两种方法差异较大。胸腿肌的嫩度不同方法存在差异,鸡种间比较建议采用胸肌为代表进行嫩度比较。     

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.     

Geographic segregation and evidence of density‐dependent changes in sex ratios in an abundant colonial waterbird

Demographic information, such as geographic segregation of sexes and sex ratio data, is needed to develop, model and evaluate conservation and management strategies for wildlife. A variety of physiological, behavioral and environmental factors can influence segregation of sexes and sex ratios, many of which originate with density‐dependent processes. Departure from 50:50 sex ratios of double‐crested cormorants (Phalacrocorax auritus) collected during control efforts in breeding and wintering areas across their eastern range of the USA were evaluated using using a Z‐test as well as Stouffer's weighted Z‐tests. In addition, a specifically‐designed randomization test was used to evaluate density‐dependent effects on primary sex ratios in cormorants from egg collections and colony nest count data over a 21‐year period. Cormorants collected from breeding colonies were strongly male‐biased, whereas cormorants collected from feeding flocks were slightly biased toward females. Cormorants were partly segregated by sex on the wintering grounds, with significantly more males found in areas with intensive channel catfish aquaculture. The null hypothesis that females produced a balanced sex ratio independent of number of nesting cormorants was rejected: more male embryos were produced during rapid population growth, whereas at maximum nesting number more female embryos were produced. Once populations stabilized, the sex ratio was more equal. This examination of sex ratios indicates that different management methods and locations result in sex‐biased culling of cormorants. Sex‐biased culling in cormorants could make population reduction efforts more efficient and reduce overall take. We suggest further research to examine density‐dependent effects on primary sex ratios documented here.