奶牛乳腺肥大细胞免疫形态学的增龄性变化

应用改良甲苯胺蓝染色法研究不同年龄奶牛乳腺肥大细胞分布特点的增龄性变化，并用Alcian bluc-safranin染色法对肥大细胞进行细胞化学分型研究。结果：青年奶牛乳腺中肥大细胞数量较少，中年奶牛乳腺中肥大细胞的数量增多，有趣的是，老年奶牛乳腺肥大的数量更多，AB-S染色显示，各年龄奶牛乳腺中只有黏膜型肥大细胞。结果表明：奶牛乳腺肥大细胞数量具有随年龄增长而增加的特点，但肥大细胞的类型没有发生改变。     

Effects of increasing days of exposure to prepartum transition diets on milk production and milk composition in dairy cows

Objective To evaluate the effects of length of exposure to prepartum transition diets on milk yield, fat and protein production. Design Prospective cohort study. The number of days that the cows were fed the prepartum transition diets was the exposure of interest. Procedures Holstein and Holstein x Jersey cows (n = 1008) were enrolled. Diets given in the far-off dry period (from end of lactation until approximately 3 weeks before expected parturition) consisted of ad libitum access to perennial ryegrass pastures. Prepartum transition diets included perennial ryegrass pasture, ryegrass silage, cereal hay, grain, grain by-product, protein meals, BioChlor(R), sodium monensin, virginiamycin or tylosin, MgSO(4), trace elements and vitamins. On a dry matter basis, these contained 16.0% crude protein, 4.2% rumen undegradable protein, and 9.9 mJ metabolisable energy/kg. Diets provided an estimated metabolisable protein balance of 286 g/day and dietary cation anion difference of -150 meq/kg dry matter. Statistical models controlled for effects of herd, calving day, breed, age and gestation period. Results Increasing length of exposure to the prepartum transition diets significantly increased the 4.0% fat- and 3.2% protein-corrected milk yield and milk-protein yield as a linear and quadratic effect. The optimal duration of exposure to the prepartum transition diets was 25 days for fat- and protein-corrected milk production and 22 days for milk protein production. Milk-fat percentage decreased significantly and linearly with increasing exposure to the prepartum transition diets; however, milk-fat yield or milk-protein percentage did not vary significantly with duration of exposure to the diets. Conclusions Increasing exposure to prepartum transition diets increased milk and milk-protein yields and decreased the milk fat-percentage, but not the milk-protein percentage or milk-fat yield. Aust Vet J 2008;86:341-351.     

IGF-Ⅰ对奶牛乳腺上皮细胞合成乳脂、乳蛋白的影响

分离培养奶牛乳腺上皮细胞,添加不同质量浓度的IGF-Ⅰ（insulin-like growth factor-Ⅰ）（0,10,100,200μg/L）刺激24h后,提取细胞总RNA,用荧光定量RT-PCR方法检测β-酪蛋白（CSN2）和乙酰辅酶A羧化酶α（ACA-CA）基因的转录水平变化。结果显示,IGF-Ⅰ能够显著促进CSN2和ACACA基因的转录,并且具有浓度依赖性。结果表明,IGF-Ⅰ可作为信号分子调节乳腺的泌乳功能。     

发育各期奶牛乳腺肥大细胞类胰蛋白酶的表达

为了研究奶牛乳腺肥大细胞的组织化学特点和分布特征,对21头发育各期奶牛乳腺进行取样,应用免疫组织化学方法（SP）,观察奶牛乳腺肥大细胞类胰蛋白酶的表达。结果显示,奶牛乳腺肥大细胞表达类胰蛋白酶,肥大细胞数量在各期有显著差异,泌乳期的乳腺肥大细胞数量明显减少（P〈0.01）,分娩后60d奶牛乳腺肥大细胞的数量最少。结果表明,肥大细胞数量随生理周期的改变而改变,静止期奶牛的局部免疫能力增强。     

苜蓿黄酮对热应激下体外培养奶牛乳腺上皮细胞凋亡的影响

本实验旨在研究苜蓿黄酮对热应激下体外培养奶牛乳腺上皮细胞的影响。将乳腺上皮细胞分成5组,每组培养基中分别含有0,25,50,75和100 μg/mL苜蓿黄酮,同时置于细胞培养箱37℃,5%CO₂培养72 h,再在 42℃恒温水浴锅中热应激1 h后返回细胞培养箱培养12 h,检测细胞活性、抗氧化指标和相关基因的表达。结果显示:1)添加25 μg/mL组的细胞活性显著高于0和50 μg/mL组(P<0.05),其他各组之间差异不显著。2)相对于0 μg/mL组,50~100 μg/mL组细胞的GSH-Px活性升高(P<0.01),LDH和MDA含量降低(P<0.01或P<0.05),而CAT活性无显著性差异。3)相对于0 μg/mL组,50和75 μg/mL组Caspase3和Socs3基因表达降低(P<0.01),25 μg/mL组P53、Stat1和Socs1基因表达升高(P<0.01或P<0.05),而Bcl-2和Fas基因表达无显著差异。综上所述,在热应激下,苜蓿黄酮能够提高体外培养奶牛乳腺上皮细胞的活性,改善抗氧化能力和抑制细胞凋亡,其中添加75 μg/mL效果较好。     

Colostrogenesis: candidate genes for IgG1 transcytosis mechanisms in primary bovine mammary epithelial cells

Bovine colostrogenesis is distinguished by the specific transfer of IgG₁ from the blood to mammary secretions. The process has been shown to be initiated by hormones and occurs during the last weeks of pregnancy when steroid concentrations of estradiol (E₂) and progesterone (P₄) are highly elevated. Rodent intestinal uptake of immunoglobulin G is mediated by a receptor termed Fc fragment of IgG, Receptor, Transporter, alpha (FcGRT) and supported by light chain Beta‐2‐Microglobulin (β2M). We hypothesized that steroid hormone treatments (E₂ and P₄) of bovine mammary epithelial cells in vitro would induce up‐regulation of IgG₁ transcytosis candidate gene mRNA expression suggesting involvement in IgG₁ transcytosis. Two different primary bovine mammary epithelial cell cultures were cultured on plastic and rat tail collagen and treated with hormonal combinations (steroids/lactogenic hormones). Evaluated mRNA components were bLactoferrin (bLf: a control), bFcGRT, β2M, and various small GTPases; the latter components are reported to direct endosomal movements in eukaryotic cells. All tested transcytosis components showed strong expression of mRNA in the cells. Expression of bFcGRT, bRab25 and bRhoB were significantly up‐regulated (p < 0.05) by steroid hormones. bRab25 and bRhoB showed increased expression by steroid treatments, but also with lactogenic hormones. Analysis for the oestrogen receptor (ER) mRNA was mostly negative, but 25% of the cultures tested exhibited weak expression, while the progesterone receptor (PR) mRNA was always detected. bRab25 and bRhoB and likely bFcGRT are potential candidate genes for IgG₁ transcytosis in bovine mammary cells.     

Effects of phenylalanine and threonine oligopeptides on milk protein synthesis in cultured bovine mammary epithelial cells

This study was conducted to investigate the effects of phenylalanine (Phe) and threonine (Thr) oligopeptides on α_s1 casein gene expression and milk protein synthesis in bovine mammary epithelial cells. Primary mammary epithelial cells were obtained from Holstein dairy cows and incubated in Dulbecco's modified Eagle's medium‐F12 medium (DMEM/F12) containing lactogenic hormones (prolactin and glucocorticoids). Free Phe (117 μg/ml) was substituted partly with peptide‐bound Phe (phenylalanylphenylalanine, phenylalanyl threonine, threonyl‐phenylalanyl‐phenylalanine) in the experimental media. After incubation with experimental medium, cells were collected for gene expression analysis and medium was collected for milk protein or amino acid determination. The results showed that peptide‐bound Phe at 10% (11.7 μg/ml) significantly enhanced α_s1 casein gene expression and milk protein synthesis as compared with equivalent amount of free Phe. When 10% Phe was replaced by phenylalanylphenylalanine, the disappearance of most essential amino acids increased significantly, and gene expression of peptide transporter 2 and some amino acid transporters was significantly enhanced. These results indicate that the Phe and Thr oligopeptides are important for milk protein synthesis, and peptide‐bound amino acids could be utilised more efficiently in milk protein synthesis than the equivalent amount of free amino acids.     

Glucose transport and milk secretion during manipulated plasma insulin and glucose concentrations and during LPS‐induced mastitis in dairy cows

In dairy cows, glucose is essential as energy source and substrate for milk constituents. The objective of this study was to investigate effects of long‐term manipulated glucose and insulin concentrations in combination with a LPS‐induced mastitis on mRNA abundance of glucose transporters and factors involved in milk composition. Focusing on direct effects of insulin and glucose without influence of periparturient endocrine adaptations, 18 dairy cows (28 ± 6 weeks of lactation) were randomly assigned to one of three infusion treatments for 56 h (six animals each). Treatments included a hyperinsulinemic hypoglycaemic clamp (HypoG), a hyperinsulinemic euglycaemic clamp (EuG) and a control group (NaCl). After 48 h of infusions, an intramammary challenge with LPS from E. coli was performed and infusions continued for additional 8 h. Mammary gland biopsies were taken before, at 48 (before LPS challenge) and at 56 h (after LPS challenge) of infusion, and mRNA abundance of genes involved in mammary gland metabolism was measured by RT‐qPCR. During the 48 h of infusions, mRNA abundance of glucose transporters GLUT1, 3, 4, 8, 12, SGLT1, 2) was not affected in HypoG, while they were downregulated in EuG. The mRNA abundance of alpha‐lactalbumin, insulin‐induced gene 1, κ‐casein and acetyl‐CoA carboxylase was downregulated in HypoG, but not affected in EuG. Contrary during the intramammary LPS challenge, most of the glucose transporters were downregulated in NaCl and HypoG, but not in EuG. The mRNA abundance of glucose transporters in the mammary gland seems not to be affected by a shortage of glucose, while enzymes and milk constituents directly depending on glucose as a substrate are immediately downregulated. During LPS‐induced mastitis in combination with hypoglycaemia, mammary gland metabolism was more aligned to save glucose for the immune system compared to a situation without limited glucose availability during EuG.     

Gene expression and hormonal regulation of adiponectin and its receptors in bovine mammary gland and mammary epithelial cells

Although the functions of adiponectin, a differentiated adipocyte‐derived hormone, in regulating glucose and fatty acid metabolism are regulated by two subtypes of adiponectin receptors (AdipoRs; AdipoR1 and AdipoR2), those in ruminants remain unclear. Therefore we examined the messenger RNA (mRNA) expression levels of adiponectin and its receptors in various bovine tissues and mammary glands among different lactation stages, and the effects of lactogenic hormones (insulin, dexamethasone and prolactin) and growth hormone (GH) on mRNA expression of the AdipoRs in cultured bovine mammary epithelial cells (BMEC). AdipoRs mRNAs were widely expressed in various bovine tissues, but adiponectin mRNA expression was significantly higher in adipose tissue than in other tissues. In the mammary gland, although adiponectin mRNA expression was significantly decreased at lactation, AdipoR1 mRNA expression was significantly higher at peak lactation than at the dry‐off stage. In BMEC, lactogenic hormones and GH upregulated AdipoR2 mRNA expression but did not change that of AdipoR1. In conclusion, adiponectin and its receptor mRNA were expressed in various bovine tissues and the adiponectin mRNA level was decreased during lactation. These results suggest that adiponectin and its receptors ware changed in mammary glands by lactation and that AdipoRs mRNA expression was regulated by different pathways in BMEC.     

Effect of interaction between leucine and acetate on the milk protein synthesis in bovine mammary epithelial cells

The interaction between Leucine (Leu) and acetate affecting milk protein synthesis in the bovine mammary epithelial cells (BMECs), and underlying the molecular mechanisms are not well understood. The objectives of this study were to investigate the effect of Leu, acetate, and their interaction on the expression of genes involved in milk protein synthesis, and JACK2/STAT5, mTOR and AMP‐activated protein kinase (AMPK) signaling pathway. The study was a 2 × 6 factorial arrangement with treatments: Leu concentration (0.45 and 1.8 mM) and acetate concentration (0, 4, 6, 8, 10, and 12 mM). The results showed that 1.8 mM Leu or 8–10 mM acetate had positive effect on ATP content, the expression of casein genes, JACK2/STAT5 and phosphorylation of mTOR pathway, but reduced AMPK phosphorylation. Leu at 1.8mM had a positive effect on the up‐regulation of acetate on ATP content, the expression of CSN1S1, CSN2, CSN3, and JACK2, the expression and phosphorylation of eukaryotic initiation factor 4E, p70 ribosomal protein S6 kinase‐1, and mTOR, but reducing AMPK phosphorylation. The results suggest that acetate, Leu, and their interaction have effect on milk protein synthesis through the JACK2/STAT5, mTOR, and AMPK pathway. Acetate addition up‐regulated the effect of Leu on milk protein synthesis, and Leu facilitated the up‐regulation of acetate on milk protein synthesis through these pathways.     

A survey of northern Victorian dairy farmers to investigate dairy calf management: colostrum feeding and management

Objectives

To describe colostrum management practices carried out in northern Victorian dairy herds and to identify weaknesses in these areas that may affect calf health and welfare by comparing the results with the current industry recommendations

Methods

A questionnaire to obtain information about colostrum management and calf‐rearing practices was sent to commercial dairy farming clients of Rochester Veterinary Practice between June and September 2013. The questionnaire consisted of a general herd overview and colostrum harvesting practices.

Results

The response rate was 39% (58/150). Many dairy producers were not meeting the current industry recommendations in the following areas: (1) time of removal calf from the dam, (2) relying on calf suckling colostrum from the dam to achieve adequate passive transfer, (3) failing to supplement calves with colostrum, (4) feeding inadequate volumes of colostrum, (5) delayed colostrum harvesting, (6) pooling of colostrum, (7) failing to objectively assess colostrum quality or relying on visual assessment and (8) storing colostrum for a prolonged periods of time at ambient temperatures.

Conclusion

The results from this survey highlight the need for greater awareness of industry standards for colostrum management and feeding hygiene.

     

牛乳腺上皮细胞的分离培养

为了对牛乳腺上皮细胞(MECs)进行分离、培养和鉴定,并研究细胞分泌功能,试验通过胶原酶消化法分离得到了牛乳腺上皮细胞,采用传代法对细胞进行纯化,对细胞标志蛋白进行免疫荧光染色鉴定,通过体外诱导和RT-PCR分析鉴定细胞的分泌功能。结果表明:分离到的牛乳腺上皮细胞具有典型乳腺上皮细胞的形态特征,表达广谱角蛋白,经诱导后可分泌β-酪蛋白。     

金黄色葡萄球菌对体外培养的奶牛乳腺上皮细胞PDGF-BB mRNA及蛋白表达的影响

为研究金黄色葡萄球菌(S.aureus)对奶牛乳腺上皮细胞(BMEC)中PDGF-BB m RNA及蛋白表达的影响,本研究采用热灭活的不同浓度的S.aureus菌液(0、105、106、108cfu/m L)作用于BMEC,分别在不同时间点(6 h、12 h、24 h、48 h)利用荧光定量PCR和western blot方法检测PDGF-BB m RNA及其蛋白的相对表达量。结果显示,不同浓度菌液处理组的PDGF-BB m RNA相对表达量随着作用时间延长表达量升高(p0.05)。6 h处理组随着菌液浓度的升高,PDGF-BB m RNA的相对表达量呈升高趋势;12 h处理组随着菌液浓度的升高,PDGF-BB m RNA的相对表达量呈降低趋势;24 h和48 h处理组105cfu/m L菌液处理组PDGF-BB m RNA相对表达量最高(p0.05)。同时,各时间点不同浓度菌液处理组PDGF-BB蛋白的相对表达量和m RNA表达基本一致。本研究表明,热灭活的S.aureus能够促进BMEC PDGF-BB m RNA和蛋白的表达。     

Proteomic analysis to unravel the effect of heat stress on gene expression and milk synthesis in bovine mammary epithelial cells

Heat stress can play a negative effect on milk yield and composition of dairy cattle, leading to immeasurable economic loss. The basic components of the mammary gland are the alveoli; these alveolar mammary epithelial cells reflect the milk producing ability of dairy cows. In this study, we exposed bovine mammary epithelial cells to heat stress and compared them to a control group using isobaric tags for relative and absolute quantitation combined with liquid chromatography coupled with tandem mass spectrometry. Compared with a control group, 104 differentially elevated proteins (>1.3‐fold) and 167 decreased proteins (<0.77‐fold) were identified in the heat treatment group. Gene Ontology analysis identified a majority of the differentially expressed proteins are associated in cell‐substrate junction assembly, catabolic processes and metabolic processes. Some of these significantly regulated proteins were related to the synthesis and secretion of milk, such as milk protein and fat. This finding was further supported by the results obtained from the reduced β‐casein expression through the system of plasminogen activator – plasminogen – plasmin and decreased fatty acid synthase could partly explain why milk fat synthesis ability of dairy cows decreased under heat stress. Our results highlight the effects of heat stress on synthesis of milk protein and fat, thus providing additional clues for further studies of heat stress on dairy milk production.     

A cell preparation of Enterococcus faecalis strain EC-12 stimulates the luminal immunoglobulin A secretion in juvenile calves

The immune system in juvenile calves is immature, so calves are susceptible to several diarrheal and respiratory diseases. Oral administration of lactic acid bacteria (LAB) is known to improve the growth performance and prevent diarrheal and respiratory diseases by stimulating the immune system in juvenile calves. Most of the immunostimulation by LAB is achieved by their cell wall components, and therefore we evaluated the immunostimulation of the cell preparation of Enterococcus faecalis strain EC-12 (EC-12) in juvenile calves in a clinical field. Twenty-nine 1-week old calves were used. Fourteen calves were administered 0.2% (w/w) of an EC-12 preparation that supplemented a milk replacer, and other calves were not supplemented. Feces and serum was collected at day 0, 7 and 49 after the administration to measure the IgA and IgG concentration. The fecal IgA concentration was increased by EC-12 administration at day 49, and the serum IgA concentration was also increased at day 7. These results suggested that oral administration of EC-12 in juvenile calves might have an immunostimulatory effect and provide earlier recovery of IgA levels in mucosal immunity.     

Establishment of an in vitro culture model to study milk production and the blood–milk barrier with bovine mammary epithelial cells

This study attempted to establish a culture model to recreate the milk production pathway in bovine mammary epithelial cells (BMECs). BMECs were isolated from Holstein cows (nonlactating, nonpregnant, and parous) and were stored by cryopreservation. To separate the apical and basolateral compartments, BMECs were cultured on a cell culture insert with a collagen gel in the presence of bovine pituitary extract and dexamethasone to induce milk production and tight junction (TJ) formation. The culture model showed the secretion of the major milk components, such as β‐casein, lactose, and triglyceride, and formed less‐permeable TJs in BMECs. Moreover, the TJs were distinctly separated from the apical and basolateral membranes. Glucose transporter‐1, which transports glucose into the cytoplasm through the basolateral membrane, localized in the lateral membrane of BMECs. Toll‐like receptor‐4, which binds to lipopolysaccharide in the alveolar lumen in mastitis, localized in the apical membrane. Beta‐casein was mainly localized near the Golgi apparatus and the apical membrane. Moreover, milk components were almost secreted into the upper chamber of the cell culture insert. These findings indicate that this model has clear cell polarity as well as in vivo and is effective to study of milk production and the blood–milk barrier in lactating BMECs.     

不同乳脂率德宏奶水牛乳腺组织中FATP1基因表达研究

为研究不同乳脂率德宏奶水牛乳腺组织中FATP1基因的表达情况,试验选取216头处于泌乳盛期的健康德宏奶水牛,分为高乳脂率组(H组)、中乳脂率组(M组)和低乳脂率组(L组),采集乳样,用乳品分析仪测定乳脂率,利用气相色谱法测定脂肪酸含量.采集乳腺组织,利用荧光定量PCR法检测FATP1基因的mRNA表达水平,并与乳脂率和...     

Bovine colostrum enriched with lyophilized bovine colostrum stimulates intestinal epithelium renewal of Holstein calves in the first days of life

Consumption of a second meal of colostrum with high quality could contribute to the intestinal epithelium development, especially if there is poor supply of colostrum just after birth. The effect of a second colostrum meal was evaluated on histomorphometry of the intestinal mucosa of newborn Holstein calves fed with high‐ and low‐quality first colostrum. Seventy‐two calves were fed with a first colostrum meal with high (HFM, close to 100 mg/ml) or low (LFM, close to 30 mg/ml) IgG concentration. At 12 hr of life, three treatments of second colostrum feeding were applied to the calves either fed high or low first colostrum: calves fed with low (LOW—close to 30 mg/ml) or high (HIGH—close to 100 mg/ml) IgG concentration; and colostrum enriched with lyophilized bovine colostrum with high IgG concentration (ENRICHED—higher than 120 mg/ml), resulting in six groups. Intestinal samples were collected after 24 and 72 hr of life. In the distal jejunum and ileum, LOW showed higher villus height than ENRICHED (p < .05). In the distal jejunum, greater villus perimeter was observed in the LOW compared to ENRICHED at 24 hr (p < .05). In ileum, LFM showed higher villus perimeter compared to HFM (p < .05). LOW showed the highest villus height‐to‐crypt depth ratio in the medium and distal jejunum and ileum, p < .05. ENRICHED and HFM showed decreased muscle layer thickness in the proximal and distal jejunum respectively (p < .05). The results reveal that the high concentration of total solids, crude protein, IgG and IGF‐I of colostrum with high quality worsened the absorptive area, but may have stimulated the activity of cell division in intestinal crypts. Considering the present results, bovine colostrum enriched with lyophilized bovine colostrum stimulates intestinal epithelium renewal of Holstein calves in the first days of life.     

Shotgun proteomics of homogenate milk reveals dynamic changes in protein abundances between colostrum,transitional, and mature milk of swine

Milk is an easily digestible source of nutrients and bioactive factors, its composition reflects the neonate’s needs, and changes from colostrum to transitional and mature milk. Our objective was to measure milk fat, lactose, total carbohydrate, and protein content in parallel with global proteome of homogenate milk samples to characterize changes across the three phases of swine lactation. Milk samples were collected from multiparous sows (n = 9) on postnatal day 0 (D0; colostrum), 3 (D3; early transitional), 7 (D7; late transitional), and 14 (D14; mature). On D3, percent fat (16 ± 2.1) and lactose (3.8 ± 0.3) were higher (P < 0.05) than on D0 (10 ± 3.9 and 1.5 ± 0.3, respectively). Levels of fat and lactose were not different between D3 and D14. Percent total protein decreased (P < 0.05) between D0 (11 ± 2.1) and D3 (5 ± 0.7), but there was no significant change in percent protein between D3 and D14. Total carbohydrates increased (P < 0.05) between D3 (944 ± 353 µg/mL) and D14 (1,150 ± 462 µg/mL). Quantitative proteomic analysis using liquid chromatography tandem mass spectrometry (LC-MS/MS) of homogenate D0, D3, and D14 milk samples (n = 6) identified 772 protein groups which corresponded to 501 individual protein-coding genes. A total of 207 high confidence proteins were detected in n = 3 sows/day. Of the high confidence proteins, 81 proteins were common among all 3 days of lactation. Among the proteins that decreased between the days (false discovery rate; FDR < 0.05) were multiple apolipoproteins and XDH which decreased between D0 to D3. Proteins that increased across the days (FDR < 0.05) were complement factors and 14-3-3 proteins (YWHAQ, YWHAE). Our data provide a good characterization of milk proteome changes that likely reflect mammary function as well as the neonate’s phase-specific developmental needs. This data may be useful in developing approaches to enhance the health and welfare of swine.