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.     

IGFs对小鼠乳腺IGFBP-5和cyclin D1表达的影响

运用荧光显微镜技术，采用小鼠乳腺组织培养及免疫荧光组化方法，对IGFs在乳腺发育和乳腺功能调控中的作用进行研究。结果表明：①IGFs能抑制各时期小鼠乳腺中IGFBP-5的表达，并且IGF—Ⅰ对小鼠乳腺中IGFBP-5表达的抑制作用强于IGF—Ⅱ，说明IGFs抑制小鼠乳腺细胞凋亡，延缓小鼠乳腺退化过程；②IGFs能促进各时期小鼠乳腺cyclin D1的表达，并且IGF—Ⅰ对小鼠乳腺cyclin D1表达的促进作用强于IGF—Ⅱ．证明IGFs可促进小鼠乳腺腺泡的发育。     

共轭亚油酸对乳腺脂类代谢和乳腺发育的作用及其机理

共轭亚油酸(Conjugated Linoleic Acid,CLA)是亚油酸具有共轭双键的一组同分异构体,由瘤胃细菌在生物氢化过程中合成,具有抗癌、抗炎症、抗动脉粥样硬化等多种生物学功能。CLA在代谢方面也有调节作用,能影响全身代谢,影响肝脏、乳腺以及富含脂肪的相关组织代谢。在此介绍了CLA的来源、结构、对动物乳腺脂类代谢和乳腺发育的作用及其机理。     

miR-9-5p对奶牛乳腺上皮细胞乳脂合成的影响

以健康的泌乳期中国荷斯坦奶牛的乳腺上皮细胞(Dairy cow mammary epithelial cells,DCMECs)为实验模型,探究mi R-9-5p对奶牛乳腺上皮细胞乳脂合成的影响。通过体外转染mi R-9-5p mimics的方式处理奶牛乳腺上皮细胞。随后采用MTT方法测定细胞增殖能力,采用western blotting技术检测乳脂合成相关信号通路蛋白SREBP1的表达情况,再采用甘油三脂检测试剂盒检测奶牛乳腺上皮细胞中甘油三脂含量的变化。实验结果显示mi R-9-5p对奶牛乳腺上皮细胞的增殖有明显的抑制作用,同时mi R-9-5p对乳脂合成相关信号通路蛋白SREBP1的表达也有明显的抑制作用,且显著抑制甘油三脂的合成,说明mi R-9-5p对奶牛乳腺上皮细胞乳脂的合成有着负调控作用。本实验试图从分子水平揭示中国荷斯坦奶牛乳品质差异的原因,进一步完善中国荷斯坦奶牛泌乳的分子机制。     

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.     

BIM 5D在项目工程施工过程中的动态管理研究

现阶段,建筑领域从业人员已经认识到建筑行业的信息化愈发重要,BIM技术在进行精细施工作业管理进程中发挥了关键的作用。建设以BIM为基础的5D施工作业进度情况的管控体系,可最大限度地辅助项目管理者进行各项决策及实施精细化的管理工作,实现项目工程进度数据信息的动态调用、进度信息比较、工程量的随时更新、资源的动态图像化管理,最大限度地提升项目管理的效率,帮助施工企业增加经济效益。     

小鼠乳腺中FGF7及其受体的表达与作用

为了从蛋白水平研究成纤维细胞生长因子7（FGF7）及其受体（KGFR）在哺乳动物乳腺发育、泌乳及退化过程中表达定位的动态变化,揭示FGF7及其受体表达变化与乳腺发育及泌乳功能的关系,本研究利用激光扫描共聚焦显微系统对昆明鼠乳腺中FGF7及其受体的表达进行了免疫荧光检测,利用体外培养不同发育时期的小鼠乳腺组织研究了FGF7在乳腺中的作用。结果表明：FGF7主要定位于乳腺导管上皮细胞以及腺泡上皮细胞外周,同时在基质中有弱的表达。KGFR主要出现在导管上皮细胞以及腺泡上皮细胞上,在乳腺发育的个别时期细胞外基质中有弱表达。FGF7在退化4d达到最高值;KGFR在泌乳4d达到最高值。除青春期和妊娠期外,添加FGF7均能显著诱导乳腺上皮细胞产生KGFR。FGF7能促进小鼠乳腺上皮细胞的增殖分化,降低乳腺上皮细胞的细胞凋亡。     

人α-乳白蛋白在奶牛乳腺上皮细胞中的表达

将人α-乳白蛋白(α-LA)0.76 kb的cDNA序列连接到PSV载体SV40启动子后,构建真核表达载体hα-LA-psv.将构建的真核表达载体转染奶牛乳腺上皮细胞,Aα-乳白蛋白的表达量约为0.85 g/L.结果表明,构建的真核表达载体能够在体外培养的牛乳腺上皮细胞中高效表达Aα-乳白蛋白.     

5S管理在肉制品企业中的应用

5S管理的概念、方法及其在肉制品企业的运用     

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.     

Infusions of casein hydrolyzates into the mammary gland disrupt tight junction integrity and induce involution in cows

Milk stasis triggers local stimuli, which make the tight junctions leak and trigger involution. The aim of the study was to test the hypothesis that casein hydrolyzates compromise tight junction integrity and dry-off milk secretion in dairy cows. Six repeated doses of casein hydrolyzates after each milking during 3 d caused drastic changes in mammary secretion and composition, which were associated with irreversible cessation of milk secretion. No such changes were recorded in the control glands that had been treated with nonhydrolyzed casein. Treatment with casein hydrolyzates disturbed tight junction integrity within 8 h (as indicated by changes in Na+ and K+ concentrations), reduced the concentrations of lactose precipitously, activated the plasmin activator-plasminogen-plasmin system, and induced the secretion of immunoglobulin type G and lactoferrin. At the end of the 3-d treatments, we stopped milking the experimental and control glands. Milk composition 19 d later was similar in the experimental and control glands and was consistent with the composition expected in fully involuted glands. We conclude that casein hydrolyzates are among the milk-borne factors that cause the disruption of tight junction integrity and induce involution in cows. The process induced by casein hydrolyzate was more rapid and synchronized than the involution induced at drying-off.     

Early mammary gland metabolic and immune responses during natural-like and forceful drying-off in high-yielding dairy cows

The present work compared metabolic and immune responses in genetically high-producing cows that produced a low amount of milk before expected involution and in cows with the same genetic potential that produced copious amounts of milk before their scheduled drying-off. Ten multiparous lactating Israeli Holstein cows producing approximately 10,500 L in the current lactation, without bacterial infection and scheduled for drying-off approximately 60 d before their expected parturition, were studied. Five of the cows that exhibited a sharp, spontaneous reduction in milk yield at the end of their lactation and produced less than ~14 L/d were defined as cows approaching natural involution (ANI), and 5 cows that produced between 25 and 35 L/d were defined as cows approaching forced involution (AFI). Three days before scheduled drying-off, milking was stopped and milk samples were collected from each quarter. After milking cessation, only modest swelling was observed in the udders of the ANI cows. In the ANI cows, lactose and fat concentrations decreased and the fat:lactose concentration ratio indicated that on d 1 and 2 fat concentrations decreased faster than lactose concentration, whereas on d 3, the rate of reduction was about the same for lactose and fat. In contrast, in AFI cows, fat concentrations increased on d 1 and the fat:lactose ratio indicated that changes in fat secretion were minor compared with those of lactose secretion. Rennet clotting time of milk after drying-off in the ANI cows increased, whereas curd firmness decreased rapidly, such that mammary secretions did not coagulate on d 3. In the AFI cows, such significant changes were observed only on d 3. The inflammatory response increased in both groups, but at each stage the increase was greater in ANI cows than in AFI cows. On d 1, the increase in leukocyte numbers in the ANI cows was made up of mononuclear cells (i.e., T lymphocytes and macrophages). In contrast, in the AFI cows, we observed a marked increase in leukocyte numbers, mainly in the form of polymorphonuclear cells. Our data indicate that the abrupt mammary involution induced in AFI cows provoked signs of distress, which were associated with neutrophilia in milk. In contrast, in the ANI cows, cessation of milking occurred without evidence of engorgement of the udder. Physiological differences in ANI and AFI cows are distinct and are reflected in the differences in the leukocyte populations in milk.     

Performance strategies affect mammary gland development in prepubertal heifers

In Brazil, the majority of dairy cattle are Holstein × Gyr (H×G). It is unknown whether excessive energy intake negatively affects their mammary development to the same extent as in purebred Holsteins. We hypothesized that mammary development of H×G heifers can be affected by dietary energy supply. We evaluated the effect of different average daily gains (ADG) achieved by feeding different amounts of a standard diet during the growing period on biometric measurements, development of mammary parenchyma (PAR) and mammary fat pad (MFP), and blood hormones. At the outset of this 84-d experiment, H×G heifers (n = 18) weighed 102.2 ± 3.4 kg and were 3 to 4 mo of age. Heifers were randomly assigned to 1 of 3 ADG programs using a completely randomized design. Treatments were high gain (HG; n = 6), where heifers were fed to gain 1 kg/d; low gain (LG; n = 6), where heifers were fed to gain 0.5 kg/d; and maintenance (MA; n = 6), where heifers were fed to gain a minimal amount of weight per day. Heifers were fed varying amounts of a single TMR to support desired BW gains. Over the 84 d, periodic biometric and blood hormone measurements were obtained. On d 84, all heifers were slaughtered and carcass and mammary samples were collected. At the end, HG heifers weighed the most (181 ± 7.5 kg), followed by LG (146 ± 7.5 kg) and MA (107 ± 7.5 kg) heifers. The ADG were near expected values and averaged 0.907, 0.500, and 0.105 ± 0.03 kg/d for HG, LG, and MA, respectively. In addition, body lengths, heart girths, and withers heights were affected by dietary treatment, with MA heifers generally being the smallest and HG heifers generally being the largest. Body condition scores differed by treatment and were highest in HG and lowest in MA heifers; in vivo subcutaneous fat thickness measurement and direct analysis of carcass composition supported this. The HG heifers had the heaviest MFP, followed by LG and then MA heifers. Amount of PAR was highest in LG heifers and was the same for HG and MA heifers. The percentage of udder mass occupied by PAR was lowest in HG heifers, differing from LG and MA heifers. Composition of MFP was not evaluated. Regarding PAR composition, no differences in ash or DM were found. On the other hand, CP concentration of PAR for HG heifers was lower than that for LG heifers, which was lower than that for MA heifers. Regarding the fat content, HG treatment was higher than LG and MA treatment, which did not differ from each other. In PAR, differences in relative abundance of genes related to both stimulation and inhibition of mammary growth were observed to depend on dietary treatment, sampling day, or both. The same can be said for most of the blood hormones that were measured in this experiment. In this experiment, high ADG achieved by feeding different amounts of a standard diet during the growing period negatively affected mammary development.     

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.     

IGFs对小鼠乳腺发育及细胞凋亡的影响

为揭示IGFs在乳腺发育和乳腺上皮细胞凋亡中的作用，运用荧光显微镜及光镜技术，采用小鼠乳腺组织培养及小鼠乳腺上皮细胞培养方法，对IGFs的作用进行了研究。结果表明，IGFs可促进小鼠乳腺导管的生长发育，并且IGF—I促小鼠乳腺导管生长发育的功能强于IGF—II；IGFs抑制泌乳期小鼠乳腺上皮细胞凋亡，并且IGF—I抑制凋亡的作用强于IGF—II。     

Hepatocyte growth factor exerts multiple biological functions on bovine mammary epithelial cells

The met proto-oncogene product Met is a member of the family of tyrosine kinase growth factor receptors, and hepatocyte growth factor/scatter factor (HGF/SF) has been identified as its only ligand. Bovine Met and HGF/SF have been recently cloned and their expression has been characterized in the mammary gland, but no data regarding the biological effects of this ligand/receptor couple in bovine mammary cells are yet available. We examined the role of HGF/SF and its receptor in a bovine mammary epithelial cell line (BME-UV). Expression of Met at the mRNA level in BME-UV mammary epithelial cells evaluated by real-time PCR was similar to the expression in MDCK cells, a widely used model for Met biology. Met expression in BME-UV at the protein level was confirmed by western blot. The analysis of some signal transductional pathways downstream from the Met receptor revealed that HGF/SF addition to BME-UV cells induced activation of the extracellular signal-regulated kinase ½ proliferative pathway and the Akt antiapoptotic pathway. The BME-UV cells treated with HGF responded with increased proliferation, cell scatter, and motility. Met activation by HGF induced degradation of the extracellular matrix and migration through matrigel coated transwells. Moreover, BME-UV cells included in a 3-dimensional matrix of collagen and treated with HGF developed tubular structures, reminiscent of the mammary gland ducts. These data indicate that HGF and Met might be important regulators of mammary gland growth, morphogenesis, and development in the bovine.     

Cellular proliferation rate and insulin-like growth factor binding protein (IGFBP)-2 and IGFBP-3 and estradiol receptor alpha expression in the mammary gland of dairy heifers naturally infected with gastrointestinal nematodes during development

Mammary ductal morphogenesis during prepuberty occurs mainly in response to insulin-like growth factor-1 (IGF-1) and estradiol stimulation. Dairy heifers infected with gastrointestinal nematodes have reduced IGF-1 levels, accompanied by reduced growth rate, delayed puberty onset, and lower parenchyma-stroma relationship in their mammary glands. Immunohistochemical studies were undertaken to determine variations in cell division rate, IGF-1 system components, and estradiol receptors (ESR) during peripubertal development in the mammary glands of antiparasitic-treated and untreated Holstein heifers naturally infected with gastrointestinal nematodes. Mammary biopsies were taken at 20, 30, 40, and 70 wk of age. Proliferating cell nuclear antigen immunolabeling, evident in nuclei, tended to be higher in the parenchyma of the glands from treated heifers than in those from untreated. Insulin-like growth factor binding proteins (IGFBP) type 2 and type 3 immunolabeling was cytoplasmic and was evident in stroma and parenchyma. The IGFBP2-labeled area was lower in treated than in untreated heifers. In the treated group, a maximal expression of this protein was seen at 40 wk of age, whereas in the untreated group the labeling remained constant. No differences were observed for IGFBP3 between treatment groups or during development. Immunolabeling for α ESR (ESR1) was evident in parenchymal nuclei and was higher in treated than in untreated heifers. In the treated group, ESR1 peaked at 30 wk of age and then decreased. These results demonstrate that the parasite burden in young heifers negatively influence mammary gland development, affecting cell division rate and parameters related to estradiol and IGF-1 signaling in the gland.