Expression of aquaporin 4 in the chicken ovary in relation to follicle development

In the mammalian ovary, aquaporins (AQPs) are thought to be involved in the regulation of fluid transport within the follicular wall and antrum formation. Data concerning the AQPs in the avian ovary is very limited. Therefore, the present study was designed to examine whether the AQP4 is present in the chicken ovary, and if so, what is its distribution in the ovarian compartment of the laying hen. Localization of AQP4 in the ovarian follicles at different stage of development was also investigated. After decapitation of hens the stroma with primordial follicles and white (1–4 mm), yellowish (4–8 mm), small yellow and the three largest yellow pre‐ovulatory follicles F3‐F1 (F3 < F2 < F1; 20–36 mm) were isolated from the ovary. The granulosa and theca layers were separated from the pre‐ovulatory follicles. The AQP4 mRNA and protein were detected in all examined ovarian compartments by the real‐time PCR and Western blot analyses, respectively. The relative expression of AQP4 was depended on follicular size and the layer of follicular wall. It was the lowest in the granulosa layer of pre‐ovulatory follicles and the highest in the ovarian stroma as well as white and yellowish follicles. Along with approaching of the largest follicle to ovulation the gradual decrease in AQP4 protein level in the granulosa layer was observed. Immunoreactivity for AQP4 was present in the granulosa and theca cells (theca interna ≥ theca externa > granulosa). The obtained results suggest that AQP4 may take part in the regulation of water transport required for follicle development in the chicken ovary.     

水牛卵泡GCs体外培养发生凋亡的生物学特征

为了解水牛卵巢卵泡中颗粒细胞（GCs）和卵母细胞在体外培养过程中发生凋亡的变化特征,通过体外培养、血清撤除法诱导水牛卵泡GCs凋亡,并应用苏木精伊红（HE）染色和DNA原位末端标记的TUNEL方法观察其病理特征;通过水牛腔前卵泡体外培养、诱导或等待其中的卵母细胞及卵泡细胞发生凋亡,然后应用透射电镜技术检查其超微结构改变。结果显示：凋亡卵泡GCs的HE染色特征包括细胞体积缩小,染色较深呈紫黑色,且多成簇分布。DNA原位末端标记检查的特征是DNA发生降解,其降解碎片呈TUNEL阳性。原始卵泡体外培养中调亡卵母细胞的超微结构表现为胞核碎裂成电子密度高的大碎片,或出现形态奇特的凋亡小体;初级卵泡中的调亡GCs,细胞内外均出现凋亡小体,细胞内还见有典型的螺层状内质网结构,其中包涵部分核质。     

重组鸡IL-2的抗球虫作用

鸡白细胞介素2(Ch IL-2)是一种重要的细胞因子,对Ch IL-2的抗球虫作用做了初步研究,分别在14日龄和21日龄给实验雏鸡胸部肌肉注射不同剂量的重组Ch IL-2蛋白,28日龄口服接种1×105个活球虫卵囊,结果表明,重组Ch IL-2蛋白能降低相对卵囊产量、减少盲肠病变、提高相对增重。其中以注射250μg重组Ch IL-2蛋白的试验组抗球虫效果最好,相对卵囊产量为23.26%,盲肠病变记分分别为0.4,相对增重率为97.80%,综合抗球虫指数(AC I)达到192.8。抗球虫效果良好,明显的优于其他试验组。     

A gene co‐expression network analysis of the candidate genes and molecular pathways associated with feather follicle traits of chicken skin

Back and thigh skin of chickens showed significant differences in the thickness and the feather follicle density and size, which are important traits for slaughtered chickens' appearance. In the present study, global gene expression profiling was conducted in the back and thigh skin of chickens using Microarray technology. The results showed that 676 genes were differentially expressed between back and thigh skin. The expression of the differentially expressed genes (DEGs), including PPP1R3C, IGF1, PTCHD1, HOXB6, FGF9, CAMK4, SHH, BMP8B, FOXN1 and PTGER2, was validated by real‐time quantitative polymerase chain reaction (RT‐qPCR), and the results were consistent with microarray results. Functional analysis revealed that the DEGs were significantly involved in cell proliferation, differentiation, apoptosis, adhesion and transport process, and the pathways were significantly mapped into the ECM‐receptor interaction, peroxisome, focal adhesion, Hedgehog and PPAR signalling pathways. Protein–protein interaction network analysis suggested that signalling pathways related to feathers morphogenesis and development, such as Wnt, FGF, MAPK, SHH and BMP signalling pathways, occupied important positions in the network. Genes involved in these signalling pathways and adhesion molecules might play a vital role in skin and feather follicle development. Further single nucleotide polymorphism (SNP) association analysis of Wnt3A showed that the AC genotype of SNP g.255361 C>A significantly increased the feather follicle density of thigh skin. Our findings may provide new insights on candidate genes and pathways related to skin and feather follicle formation of chickens.     

Heat stress decreased hair follicle population in rex rabbits

The aim of this study was conducted to investigate the effect of heat stress on the hair follicle population and related signalling pathways in rex rabbits. Forty‐eight rabbits were randomly divided into two groups: one group in a high ambient environment (32 ± 2°C, heat stress) and the other group with normal temperature (20 ± 2°C, control). The results show that heat stress decreased the body weight gain and feed conversion rate, rabbit hair length and hair follicle density (p < 0.05). Besides, heat stress suppressed the gene expression of noggin, insulin‐like growth factor 1 (IGF‐1) and IGF‐1 receptor and protein expression of phosphorylated mechanistic target of rapamycin (mTOR) in rabbit skin (p < 0.05), while stimulated significantly the gene expression of bone morphogenetic protein 2 (BMP2) and BMP4 (p < 0.05). Heat exposure did not alter significantly the gene expression of alkaline phosphatase, versican and hepatocyte growth factor compared with the control (p > 0.05). In conclusion, noggin‐BMP, IGF‐1 and mTOR signalling pathways may be associated with the process of heat stress‐repressing hair follicle development.     

武夷山市武夫镇鸡球虫病流行病学调查

对武夷山市武夫镇2014年采集的95份鸡粪便涂片镜检,结合临诊症状、病理剖检情况、问卷调查综合判断,表明该病在武夫镇不同时间、不同鸡场、不同日龄鸡群中均有不同程度的感染。     

Methionine deficiency and its hydroxy analogue influence chicken intestinal 3-dimensional organoid development

Methionine and its hydroxy analogue(MHA)have been shown to benefit mouse intestinal regeneration.The intestinal organoid is a good model that directly reflects the impact of certain nutrients or chemicals on intestinal development.Here,we aimed to establish a chicken intestinal organoid culture method first and then use the model to explore the influence of methionine deficiency and MHA on intestinal organoid development.The results showed that 125-mm cell strainer exhibited the highest efficiency for chicken embryo crypt harvesting.We found that transforming growth factor-b inhibitor(A8301)supplementation promoted enterocyte differentiation at the expense of the proliferation of intestinal stem cells(ISC).The mitogen-activated protein kinase p38 inhibitor(SB202190)promoted intestinal organoid formation and enterocyte differentiation but suppressed the differentiation of enteroendocrine cells,goblet cells and Paneth cells.However,the suppression of enteroendocrine cell and Paneth cell differentiation by SB202190 was alleviated at the presence of A8301.The glycogen synthase kinase 3 inhibitor(CHIR99021),valproic acid(VPA)alone and their combination promoted chicken intestinal organoid formation and enterocyte differentiation at the expense of the expression of Paneth cells and goblet cells.Chicken serum significantly improved organoid formation,especially in the presence of A8301,SB202190,CHIR99021,and VPA,but inhibited the differentiation of Paneth cells and enteroendocrine cells.Chicken serum at a concentration of 0.25%meets the requirement of chicken intestinal organoid development,and the beneficial effect of chicken serum on chicken intestinal organoid culture could not be replaced by fetal bovine serum and insulin-like growth factor-1.Moreover,commercial mouse organoid culture medium supplemented with A8301,SB202190,CHIR99021,VPA,and chicken serum promotes chicken organoid budding.Based on the chicken intestinal organoid model,we found that methionine deficiency mimicked by cycloleucine suppressed organoid formation and organoid size,and this effect was reinforced with increased cycloleucine concentrations.Methionine hydroxy analogue promoted regeneration of ISC but decreased cell differentiation compared with the results obtained with L-methionine.In conclusion,our results provide a potentially excellent guideline for chicken intestinal organoid culture and insights into methionine function in crypt development.     

牛腔前卵泡体外培养前后质量评定

采用台盼蓝染色、相差显微镜观察以及透射电镜方法对体外培养前后牛腔前卵泡活力进行评定。结果表明,台盼蓝染色和相差显微镜观察检测卵泡活力有一定的误检率,超微结构检测能够客观、真实地反映腔前卵泡健康状况,可作为评定腔前卵泡培养系统优劣的一个可靠手段。在实际应用中,相差显微镜观察与超微结构评定相结合可发挥良好作用。     

高效液相色谱法检测鸡肉中替米考星药物残留的检测与分析

本研究应用高效液相色谱法对来自市场中64批次鸡肉样品进行替米考星药物残留的检测。结果表明,替米考星药物残留物在鸡肉中的平均回收率为70.5%～92.3%。显示该方法具有灵敏、快速的特点,符合现行兽药残留分析的要求。替米考星药物残留有一定检出率,相关部门应加强监管。     

鸡致病性大肠杆菌耐药性的研究进展

大肠杆菌是一种常见致病菌,近年来,大肠杆菌的耐药株不断增多,特别是多重耐药株的大量出现,使其治疗变得十分困难。本文对鸡致病性大肠杆菌耐药现状以及产生耐药性机制的研究作以综述,以期为进一步研究鸡大肠杆菌病的综合防治提供依据。     

Melatonin supplement induced the hair follicle development in offspring rex rabbits

Previous studies have shown that the administration of melatonin (MT) to early post-natal fur-bearing animals increases the numbers of hair follicles (HFs). In this study, the effect of maternal MT supplementation on the HF development in offspring was investigated. To explore the potential underlying mechanisms, the expression of the melatonin receptor 1A (MTNR1A) gene was determined in the offspring. The Rex rabbit was the animal model, and 79 same-day-pregnancy females were randomly assigned to either a control (n = 39) or MT treatment (n = 40) group, and 10 mg MT microcapsules was implanted at the base of the neck of rabbits in the treatment group. Skin, lung, liver, muscle, kidney, heart and duodenum samples were collected from the newborn rabbits. The results showed that MT improved fur quality in the offspring rabbits by reducing the diameter of primary and secondary HFs, and increasing the HF population. MT did not affect the reproductive performance of female rabbits, and it did affect the blood levels of thyroid-stimulating hormone, prolactin and MT. In the offspring rabbits, MT significantly stimulated MTNR1A gene expression in the skin and heart (p < .01), whereas MTNR1A gene expression was significantly suppressed in the liver and kidney (p < .05). These results revealed that maternal MT supplementation increased HF density, reduced hairiness and improved the fur quality in Rex rabbit offspring. Maternal MT supply may modulate the responses of HFs in the offspring by upregulating the expression of MTNR1A in the skin. In this study, implantation of low-dose MT did not affect the reproductive performance of female rabbits or on the growth of their offspring.     

Influence of nitric oxide on in vitro growth, survival, steroidogenesis, and apoptosis of follicle stimulating hormone stimulated buffalo (Bubalus bubalis) preantral follicles

Effect of sodium nitroprusside (SNP), a nitric oxide (NO) donor, on in vitro survival, growth, steroidogenesis, and apoptosis of buffalo preantral follicles (PFs) was investigated. PFs (200~250 µm) were isolated by micro-dissection and cultured in 0 (control), 10^-3, 10^-5, 10^-7, and 10^-9 M SNP. To examine the reversible effect of SNP, PFs were cultured with 10^-5 M SNP + 1 mM N^ω-nitro-L-arginine methyl ester (L-NAME) or 1.0 µg hemoglobin (Hb). The results showed that greater concentrations of SNP (10^-3, 10^-5, 10^-7 M) inhibited (p < 0.05) FSH-induced survival, growth, antrum formation, estradiol production, and oocyte apoptosis in a dose-dependent manner. However, a lower dose of SNP (10^-9 M) significantly stimulated (p < 0.05) the survival, growth, antrum formation, follicular oocyte maturation, and stimulated progesterone secretion compared to the control. A combination of SNP + L-NAME promoted the inhibitor effect of SNP while a SNP + Hb combination reversed this effect. Nitrate and nitrite concentrations in the culture medium increased (p < 0.05) in a dose-dependent manner according to SNP concentration in the culture medium. At higher concentrations, SNP had a cytotoxic effect leading to follicular oocyte apoptosis whereas lower concentrations have stimulatory effects. In conclusion, NO exerts a dual effect on its development of buffalo PFs depending on the concentration in the culture medium.     

Status of autophagy,lysosome activity and apoptosis during corpus luteum regression in cattle

Corpus luteum (CL) regression is required during the estrous cycle. During CL regression, luteal cells stop producing progesterone and are degraded by apoptosis. However, the detailed mechanism of CL regression in cattle has not been fully elucidated. The aim of this study was to evaluate autophagy, lysosome activity, and apoptosis during CL regression in cattle. The expression of autophagy-related genes (LC3α, LC3β, Atg3, and Atg7) and the protein LC3-II was significantly higher in the late CL than in the mid CL. In addition, autophagy activity was significantly increased in the late CL. Moreover, gene expression of the autophagy inhibitor mammalian target of rapamycin (mTOR) was significantly lower in the late CL than in the mid CL. Lysosome activation and expression of cathepsin-related genes (CTSB, CTSD, and CTSZ) showed significant increases in the late CL and were associated with an increase in cathepsin B protein. In addition, mRNA expression and activity of caspase 3 (CASP3), an apoptotic enzyme, were significantly higher in the late CL than in the mid CL. These results suggest simultaneous upregulation of autophagy-related factors, lysosomal enzymes and apoptotic mediators, which are involved in regression of the bovine CL.     

鸡EX-FABP基因PCR-SSCP分析

以AA白鸡、广西鸡、E1矮小鸡品系为试验材料,采用PCR-SSCP技术对鸡细胞外脂肪酸结合蛋白基因(EX-FABP)的2个片段进行PCR扩增,分析了EX-FABP基因在3个鸡品系的多态性。结果表明:EX-FABP基因在2对引物扩增片段中均存在PCR-SSCP多态性。对于引物1扩增片段,3个鸡品系均检测到BB基因型,AA基因型只出现在AA白鸡中;而且B等位基因频率在3个鸡品系中均明显高于A等位基因频率。对于引物2扩增片段,3个鸡品系均检测到HH和Hh基因型,hh基因型只出现在E1矮小鸡中;H等位基因频率在3个品系中均明显高于h等位基因频率。引物1和引物2的多态性片段测序分析表明,EX-FABP基因第641位点和645位点分别发生了单碱基的转换(G-A和T-C),第3264位点发生了转换(T-C)。     

Regulation of primordial follicle formation,dormancy, and activation in mice

In female reproduction, the oocyte number is limited after birth. To achieve a continuous ovulatory cycle, oocytes are stored in primordial follicles. Therefore, the regulation of primordial follicle dormancy and activation is important for reproductive sustainability, and its collapse leads to premature ovarian insufficiency. In this review, we summarize primordial follicle development and the molecular mechanisms underlying primordial follicle maintenance and activation in mice. We also overview the mechanisms discovered through in vitro culture of functional oocytes, including the establishment of primordial follicle induction by environmental factors, which revealed the importance of hypoxia and compression by the extra cellular matrix (ECM) for primordial follicle maintenance in vivo.     

Phosphorothioate oligodeoxyribonucleotides induce in vitro proliferation of chicken B-cells

The study aimed to evaluate short synthetic oligodeoxyribonucleotides (ODN) as inducers of proliferation of chicken peripheral blood mononuclear cells (PBMC) and to identify the proliferating cells. A panel of different ODN; with phosphodiester and/or phosphorothioate backbone, with and without CpG-motifs, was therefore assessed for in vitro induction of proliferation.Six complete phosphorothioate ODN induced proliferation of PBMC while the complete phosphodiester or chimeric phosphodiester/phosphorohiate ODN did not. Moreover, CpG-motifs were not essential for induction of proliferation as responses to CpG-ODN were similar to those of their GpC controls. Two stimulatory phosphorothioate ODN were also used in phosphodiester form. In this comparison, only the phosphorothioate ODN were active despite the identical nucleotide sequences of their phosphodiester counterparts.In order to deliver DNA to the cytoplasm and decrease degradation of ODN by nucleases, stimulating as well as inactive ODN were treated with lipofectin prior to induction. However, proliferative responses were not influenced by lipofectin treatment and in analogy, none of the inactive ODN induced proliferation after lipofectin treatment.Among PBMC, ODN-responding cells were identified as predominantly Bu-1, immunoglobulin and major histocompatibility complex class II expressing cells, while CD3 expressing cells were not responding. Using magnetic cell separation of Bu-1 expressing cells prior to culture it was found that Bu-1 depleted cells did not proliferate upon ODN stimulation while the Bu-1 enriched cells were able to proliferate upon this stimulus.Taken together, among ODN in the present panel, only phosphorothioate ODN induced proliferation of PBMC. Responses were induced regardless of the presence of CpG-motifs and were not influenced by addition of lipofectin. Amid the chicken PBMC, predominantly cells of a B-cell phenotype proliferated in response to ODN stimulation and they were able to respond to this stimulus without the presence of other cell types.     

海兰褐鸡血清淀粉酶同工酶

采用聚丙烯酰胺凝胶电泳法对５５只海兰褐鸡血清淀粉酶同工酶进行研究。结果：①血清淀粉酶有ＡＭＹ１，ＡＭＹ２和ＡＭＹ３三种同工酶；②ＡＭＹ２同工酶存在ＡＭＹ２ＡＡ，ＡＭＹ２ＡＢ和ＡＭＹ２Ｅ８三种表型，其表型频率分别为０．１８，０．３１和０．５１；③各同工酶区带的相对迁移率为：ＡＭＹ１１０．２１％，ＡＭＹ２ＡＡ８．１６％，ＡＭＹ２ＢＢ６．６３％，ＡＭＹ３２．０４％。     

高原地区矮小鸡与藏鸡屠宰试验

通过对在高原地区10只成年矮小鸡和10只成年藏鸡屠宰测定,分析结果显示:平均体重矮小鸡比藏鸡高2．2％;藏鸡公鸡全净膛率为53．9％,矮小鸡公鸡全净膛率为64．9％,差异极显著;藏鸡母鸡全净膛率为58．1％,矮小鸡母鸡全净膛率为51．5％,差异极显著;藏鸡公鸡半净膛率为74．2％,矮小鸡公鸡半净膛率为81．5％,差异显著;藏鸡母鸡半净膛率为77．1％,矮小鸡母鸡半净膛率为66．2％,差异极显著。经测定说明矮小鸡的屠宰率高于藏鸡。