GLGI技术鉴定奶山羊乳腺中的к-酪蛋白基因

采用GLGI技术扩增本实验室构建的Long-SAGE标签库中的к-酪蛋白基因.使用SMARTTM RACE cDNA Ampliication Kit通过RT-PCR把奶山羊乳腺组织中的rnRNA逆转录成cDNA.分别用3′RACE和5′ACE扩增к-酪蛋白基因的3′端和5′端.扩增产物经TA克隆后转化大肠杆菌,随机挑取阳性克隆后进行测序分析.通过序列比对、拼接,获得全长的к-酪蛋白基因.应用GLGI技术成功获得了完整的奶山羊乳腺CSN3序列,为CSN3结构分析和功能研究奠定了重要基础.     

奶山羊青春期乳腺发育重要基因RelA的全长克隆

从本实验室已建立的关中奶山羊7个时期乳腺Long-SAGE标签库中筛选出在青春期高表达的RelA基因,用RACE方法克隆并获得关中奶山羊RelA基因的全长,将全长序列与NCBI GenBank中人(H.sapiens)和牛(Bos taurus)数据库进行同源性比对,证实用所选EST标签引物克隆出的基因全长是奶山羊RelA基因.     

奶山羊乳腺发育的超微结构研究

根据奶山羊乳腺发育特点及生长阶段的不同分为青春期、妊娠期、泌乳期及退化期,应用透射电镜对健康中国关中奶山羊乳腺发育不同时期的超微结构特点和形态变化进行了研究与讨论.     

奶山羊乳腺中胰岛素样生长因子及其受体的表达与作用

为研究胰岛素样生长因子及胰岛素样生长因子受体在奶山羊乳腺组织青春期、妊娠期、泌乳期及退化期表达的差异及变化,探索其在奶山羊乳腺发育过程中的作用,根据奶山羊乳腺发育特点及生长阶段不同分为青春期、妊娠期、泌乳期及退化期,采用激光共聚焦显微镜检测胰岛素样生长因子在奶山羊乳腺各生长阶段的表达及定位,通过定量分析来比较胰岛素样生长因子在奶山羊乳腺各生长阶段表达的差异,描述其在奶山羊乳腺发育过程中的作用.结果显示:胰岛素样生长因子及胰岛素样生长因子受体在奶山羊乳腺法如各时期均有表达,表达水平与乳腺发育密切相关.     

奶山羊和荷斯坦奶牛乳腺组织mRNA差异显示体系建立

利用mRNA差异显示技术对关中奶山羊和荷斯坦奶牛泌乳末期乳腺组织进行差异表达基因分析,为进一步进行奶山羊泌乳分子生物学研究及筛选克隆差异表达基因、揭示膻味脂肪酸调控的分子机理奠定基础.研究通过对各影响因素进行单因素分析,建立了适合奶山羊和荷斯坦奶牛的乳腺差异显示分析体系,得到的最佳体系.在20 μL DDRT-PCRR反应总体系中各组分的添加量分别为:13.0 μL DEPC-H2O;1.0 μL 10×PCK Buffer(加Mg2+);1.5 μL dNTPs(各2.5 mmol/L);1.5 μL锚定引物H-T11M(20 μmol/L);1.5 μL随机引物(10 μmol/L);1.0 μL cDNA;0.5 μL Taq DNA Polymerase(2 U/μL).     

奶山羊乳腺发育过程中腺苷酸质量分数及能荷的变化

探讨了奶山羊乳腺发育和泌乳过程中能量代谢变化规律和特点.分别在奶山羊乳腺发育的4个时期青春期、妊娠期、泌乳期及退化期采集乳腺组织.应用高效液相色谱测定各时期的腺苷酸质量分数并计算出各时期能荷:用总ATP酶活性测定试剂盒测定各时期的ATP酶活性.结果表明,青春期和妊娠期腺苷酸质量分数和ATP酶活性呈缓慢递增趋势,泌乳启动后ATP酶活性急剧增加,腺苷酸质量分数增加,ADP、AMP比例升高.在泌乳高峰期,AMP达到峰值,能荷值最低.泌乳高峰期过后ATP酶活性下降,腺苷酸质量分数减少,AMP比例急剧减少,能荷值恢复到正常水平.在乳腺发育过程中,泌乳启动后乳腺ATP酶活性急剧增加,腺苷酸质量分数增加;泌乳高峰期过后ATP酶活性下降,腺苷酸质量分数减少.     

奶山羊泌乳激素及其受体在乳腺发育过程中的变化规律及作用

探讨奶山羊乳腺发育过程中泌乳激素(E2,P和PRL)及其受体(ERα,PR和PRLR)的变化规律及作用.分别在关中奶山羊乳腺发育的青春期、妊娠期、泌乳期及退化期,采集血清并用放射性免疫学方法测定各激素浓度,采集乳腺组织并用免疫荧光和real-time PCR检测各激素受体表达量.青春期和妊娠早期E2,P和PRL缓慢上升,妊娠晚期E2和P达峰值,泌乳启动PRL高峰,泌乳高峰过后各激素均回复正常水平.ERα和PRLR青春期开始缓慢上升,泌乳启动后ERα达到高峰,而泌乳期PRLR维持在高水平,并在退化期达到表达高峰.青春期PR较妊娠期高,妊娠后期又开始升高,退化期下降.E2与ERα,E2与P及与ERα之间正相关,其余的激素和受体之间相关性不显著.     

奶山羊乳腺细胞凋亡及瘦素对凋亡的影响

为了阐明奶山羊乳腺正常发育过程中的细胞凋亡情况及瘦素对乳腺凋亡的影响,以中国关中奶山羊乳腺为材料,采用TUNEL法、透射电镜法及免疫组化方法对奶山羊乳腺发育全程的凋亡情况及瘦素对凋亡的影响进行检测。结果表明,青春期、妊娠期和泌乳初期细胞凋亡量维持在很低水平,泌乳晚期和退化期随着泌乳的结束,乳腺导管和腺泡结构开始瓦解,大量细胞发生凋亡,退化7 d达到最大值,退化21 d后乳腺已经基本恢复到青春期状态,凋亡细胞量随之减少。将退化7 d的乳腺组织分别在含有瘦素和不含瘦素的培养基中培养,培养后的组织分别用TUNEL法检测细胞凋亡,结果显示不加瘦素培养的组织形态保持较好,凋亡细胞较少;加瘦素培养的组织导管和腺泡结构已经开始发生解体,组织中有较多的凋亡细胞。结论:山羊乳腺发育不同时期细胞凋亡同结构和功能发育之间相互联系;瘦素在退化期乳腺组织中可明显诱导细胞凋亡。     

高山红景天中糖基转移酶家族cDNA全长基因的克隆

为了获得高山红景天中红景天苷生物合成关键酶-- UDP- 糖基转移酶家族基因的全长cDNA 序列,采用在线简并引物设计软件结合3′-RACE 技术获得两个糖基转移酶基因的3′端部分序列,进一步采用不同的5′-RACE试剂盒扩增两个基因的5′端序列,结果显示利用Takara 公司的试剂盒扩增可以得到cDNA全长序列(GenBank登录号为EF508689 和EU567325),而采用Invitrogen 公司的试剂盒得到的序列不包括全部开放读码框。实验证明,利用Takara 公司的SMARTerTM RACE cDNA Amplification Kit 克隆获得基因全长序列的比率更高。     

奶山羊乳腺中瘦素的信号转导通路

瘦素是影响乳腺发育和泌乳的自分泌和旁分泌因子,为阐明瘦素在奶山羊乳腺发育、泌乳及退化过程中的信号转导通路,本实验以中国关中奶山羊为实验动物,取妊娠90 d、泌乳60 d和退化7 d的山羊乳腺组织体外无血清培养48 h,对照组不添加瘦素,提取乳腺总蛋白,采用Western Blot方法进行系统研究.结果表明:妊娠、泌乳及退化各时期中均有无活性的非磷化MAPK.STAT3和STAT5存在.妊娠期瘦素专一性地诱导JAK-MAPK信号转导通路中p42MAPK/p44MAPK磷酸化;泌乳期瘦素主要诱导信号转导子和转录激活子STAT5的磷酸化;退化期,瘦素专一性诱导STAT3磷酸化.     

奶牛乳腺中瘦素及其受体的表达与定位

为探讨瘦素在奶牛乳腺发育、泌乳及退化各时期的表达变化规律及在乳腺组织中的具体位置,采用免疫印迹(western blot-ting)技术及激光共聚焦技术检测奶牛乳腺组织中瘦素及瘦素受体(OB-Rb)的表达变化及其定位。结果表明,瘦素在青春期表达量较高,在泌乳期表达量最低。瘦素受体在青春期和妊娠期表达量较高,泌乳期表达量相对较低,退化期逐渐恢复到妊娠期水平。     

Role of prolactin, growth hormone and insulin-like growth factor 1 in mammary gland involution in the dairy cow

Bovine mammary involution, an important process for subsequent lactations, is characterized by loss of epithelial cells by apoptosis, but its hormonal regulation is still not well defined. Prolactin (PRL) and growth hormone (GH) play a specific role on rat mammary gland apoptosis, through insulin-like growth factor 1 (IGF-1) and the IGF binding protein (IGFBP) system. The purpose of our investigation was to determine the possible role of PRL, GH, and IGF-1 on cell survival and on IGFBP-5 expression in the bovine mammary gland. Mammary gland explants were cultured in the presence of cortisol, 17beta-estradiol, progesterone, insulin, PRL, GH, and IGF-1 and with the same treatment but without PRL, GH or IGF-1, respectively. After 24 h of culture, we determined the level of apoptosis through evaluation of DNA laddering in the oligonucleosomal fraction and examined IGFBP-5 messenger RNA (mRNA) expression. The results show a high level of DNA laddering and an increase in IGFBP-5 mRNA content in mammary explants cultured in the absence of PRL, GH, or IGF-I with respect to explants treated with all hormones. Moreover, explants cultured in presence of PRL, GH, or IGF-I show a low level of DNA laddering and IGFBP-5 expression with respect to explants cultured without any hormones. These data demonstrate a relationship between levels of apoptosis and IGFBP-5 mRNA expression in the bovine mammary gland and confirm the involvement of this binding protein programmed cell death and its relationship with the main lactogenic hormones.     

王不留行促进奶牛乳腺泌乳的信号转导途径

应用Western blot检测王不留行水浸提液作用后,乳腺上皮细胞泌乳信号转导基因P-AKT1、cyclinD1、PRLR、P-STAT5a和AMPK、GULT1的变化,结果表明:中药王不留行对P-AKT1、cyclinD1、PRLR、P-STAT5a和AMPK、GULT1基因表达具有一定作用,可能通过mTOR信号转导途径促进乳腺上皮细胞的增殖;通过激活PRLR从而激活Stat5a泌乳信号转导途径激活其下游β-酪蛋白基因的表达;且可能通过激活AMPK而促进GLUT1表达的增强,从而增加细胞对葡萄糖的摄取能力促进乳糖的合成。     

Bovine somatotropin administration to dairy goats in late lactation: effects on mammary gland function,composition and morphology

We investigated the effects of bovine somatotropin (bST) on mammary gland function and composition in the declining phase of lactation in goats. Sixteen Saanen goats, 180 +/- 11 days in milk (DIM), were divided equally into control and treated groups. The treated group received 120 mg/2 wk of slow-release bST for three cycles. Milk yield, milk composition, milk clotting measures, and plasmin-plasminogen activator activities were recorded weekly. Milk Na and K were determined in individual milk samples collected weekly during the third cycle. Blood samples were collected weekly during the second cycle and the plasma analyzed for nonesterified fatty acids (NEFA), glucose, and urea. At the end of the 6 wk, three goats from each group were slaughtered, and the udders were removed. Mammary gland weight, composition, and total DNA content were determined. The histological effects of bST on mammary tissue were investigated. The analyzed parameters included numbers of alveoli, corpora amylacea, apoptotic cells, and laminin fibronectin distribution and localization. An extensive morphological analysis on the epithelial and stromal components was performed. Milk yield was significantly higher in the treated group, fat content was not affected, but protein and nonprotein nitrogen were lower in treated goats milk. Treatment with bST did not influence milk pH but reduced coagulation time. Plasmin and plasminogen activator activities were not affected. Milk K levels were higher and the Na/K ratio was lower in treated animals. Plasma glucose, NEFA, and urea were unaffected. Mammary gland weight and total DNA were higher in treated than control animals, suggesting that with advancing lactation bST treatment maintains cells. Fat, protein, and collagen content of the mammary tissue did not differ between the groups. Treatment with bST significantly increased the number of lactating alveoli (LA) and significantly reduced the number of regressing alveoli (RA) and corpora amylacea, both within and outside the alveolar lumen. Laminin and fibronectin localization were not affected, and very few apoptotic cells were found in both treated and control samples. Our findings suggest that bST administration to dairy goats in late lactation can modulate mammary gland activity and improve lactation persistency; this is associated with maintained total mammary parenchyma weight and lactating alveoli.     

Technical note: Mammary gland ultrasonography to evaluate mammary parenchymal composition in prepubertal heifers

Bovine mammary gland development studies are often terminal or involve invasive biopsy procedures. Therefore, noninvasive means of assessing mammary development should be considered as alternative methods in live animals. The objective was to test if mammary ultrasonography can be used as a noninvasive way to estimate mammary parenchyma (PAR) composition in prepubertal dairy heifers with different average daily body weight gains. In the 84 d preceding, the ultrasound exam heifers were maintained in 1 of 3 treatment groups. Individual heifers were fed a high gain (1 kg/d; n = 6), low gain (0.5 kg/d, n = 6), or maintenance (n = 6) treatment diet. To achieve desired body weight gains, heifers were fed differing amounts of the same silage-based diet. Mammary glands of 18 crossbred heifers Holstein:Gyr underwent a single mammary ultrasound exam immediately before heifer slaughter, which took place when heifers weighed 142.0 ± 8.0 kg and were 200 d old. The 4 mammary glands of each heifer were evaluated using a real-time B-mode ultrasound machine equipped with a 6.5-MHz micro-convex transducer. Digital images (8-bit) of glands were obtained and PAR was identified within gland. Average pixel values per unit of PAR area were determined for each gland and analyzed at the level of heifer. Pixel results were interpreted on the basis that lower average pixel values reflect PAR with relatively high amounts of protein as opposed to fat. To help validate that the pixel value within PAR is associated with composition of PAR, pixel findings were compared with histological [number of adipocytes in PAR (Nad) and epithelial area in PAR (Ep)] and biochemical [percent crude protein in PAR (%CP), percent ether extract in PAR (%EE), PAR weight (WPAR), and mammary fat pad weight (WFAT)] composition of PAR in these same heifers. Within PAR, %EE and WFAT were positively correlated with pixel values, whereas %CP, Ep, and Nad were negatively correlated. Parenchyma weight did not correlate with pixel values. Regression analyses (fixed effect log-pixel value; random effect treatment) were used to estimate Nad, Ep, %CP, %EE, WPAR, and WFAT. Sensitivity analysis of regression equations revealed that accuracy of tested equations ranged from 0.77 to 0.93 and precision ranged from 0.56 to 0.82. Concordance correlation coefficients of the equations ranged from 0.41 to 0.76. In conclusion, ultrasonography of PAR can accurately measure and predict PAR composition in prepubertal dairy heifers growing at various rates of gain.     

Invited review: Metabolic challenges and adaptation during different functional stages of the mammary gland in dairy cows: Perspectives for sustainable milk production

Milk production of dairy cows has increased markedly during recent decades and continues to increase further. The evolutionarily conserved direction of nutrients to the mammary gland immediately after calving provided the basis for successful selective breeding toward higher performance. Considerable variation in adaptive responses toward energy and nutrient shortages exists; however, this variation in adaptability recently gained interest for identifying more metabolically robust dairy cows. Metabolic challenges during periods of high milk production considerably affect the immune system, reproductive performance, and product quality as well as animal welfare. Moreover, growing consumer concerns need to be taken into consideration because the public perception of industrialized dairy cow farming, the high dependency on feed sources suitable for human nutrition, and the apparently abundant use of antibiotics may affect the sales of dairy products. Breeding for high yield continues, but the metabolic challenges increasingly come close to the adaptational limits of meeting the mammary gland's requirements. The aim of the present review is to elucidate metabolic challenges and adaptational limitations at different functional stages of the mammary gland in dairy cows. From the challenges and adaptational limitations, we derive perspectives for sustainable milk production. Based on previous research, we highlight the importance of metabolic plasticity in adaptation mechanisms at different functional stages of the mammary gland. Metabolic adaptation and plasticity change among developing, nonlactating, remodeling, and lactational stages of the mammary gland. A higher metabolic plasticity in early-lactating dairy cows could be indicative of resilience, and a high performance level without an extraordinary occurrence of health disorders can be achieved.     

Regulation of fluid flow through the mammary gland of dairy cows and its effect on milk production: a systematic review

Dairy milk consists of more than 85% water. Therefore, understanding the regulation of fluid absorption in the mammary gland is relevant to improving milk production. In recent decades, studies using different approaches, including blood flow, transmembrane fluid flow, tight junction, fluid flow of the paracellular pathway and functional mammary epithelial cell state, have been conducted aiming to investigate how mammary gland fluid absorption is regulated. However, the relationship between regulation mechanisms of fluid flow and milk production has not been studied systematically. The present review summarizes a series of key milk yield regulatory factors mediated by whole‐mammary fluid flow, including milk, mammary blood flow, blood/tissue fluid–cell fluid flow and cell–alveolus fluid flow. Whole‐mammary fluid flow regulates milk production by altering transporter activity, ion channels, local microcirculation‐related factors, driving force of fluid transport (osmotic pressure or electrochemical gradient), cellular connection state and a cell volume sensitive mechanism. In addition, whole‐mammary fluid flow plays important roles in milk synthesis and secretion. Knowledge gained from fluid flow‐mediated regulatory mechanisms of the dairy mammary gland will lead to a fundamental understanding of lactation biology and will be beneficial for the improvement of dairy productivity. © 2017 Society of Chemical Industry     

Effects of photoperiod modulation and melatonin feeding around drying-off on bovine mammary gland involution

The risk for a dairy cow to acquire new intramammary infections is high during the transition from lactation to the dry period, because of udder engorgement and altered immune functions. Once the gland is fully involuted, it becomes much more resistant to intramammary infections. Therefore, strategies to depress milk yield before drying-off and accelerate the involution process after drying-off could be beneficial for udder health. The objective of this study was to assess the effect of photoperiod manipulation and melatonin feeding from 14 d before to 14 d after drying-off on the speed of the involution process. Thirty Holstein cows in late lactation were randomly allocated to one of the following treatments: (1) a long-day photoperiod (16 h of light: 8 h of darkness), (2) a short-day photoperiod (8 h of light: 16 h of darkness), and (3) a long-day photoperiod supplemented by melatonin feeding (4 mg/kg of body weight). Milk and blood samples were collected on d ?26, ?19, ?12, ?5, ?1, 1, 3, 5, 7, 10, and 14 relative to the last milking to determine concentrations of mammary gland involution markers and serum prolactin. Additional blood samples were taken around milking on d ?15, before the start of the treatments, and on d ?1, before drying-off, to evaluate the treatment effects on milking-induced prolactin release. The short-day photoperiod slightly decreased milk production and basal prolactin secretion during the dry period. The milking-induced prolactin surge was smaller on d ?1 than on d ?15 regardless of the treatments. Lactoferrin concentration, somatic cell count, and BSA concentration as well as matrix metalloproteinase-2 and -9 activities increased in mammary secretions during the first 2 wk of the dry period, whereas milk citrate concentration and the citrate:lactoferrin molar ratio decreased. The rates of change of these parameters were not significantly affected by the treatments. The long-day photoperiod supplemented by melatonin feeding did not affect milk production, prolactin secretion, or mammary gland involution. Under the conditions in this study, photoperiod modulation and melatonin feeding did not appear to affect the rate of mammary gland involution.