【作者】 吴显实；

【导师】 黄克和；

【作者基本信息】 南京农业大学 ， 临床兽医学， 2009， 博士

【摘要】 硒是动物和人所必需的微量元素之一,具有增强机体抗氧化能力、免疫力,提高繁殖性能,减少糖尿病、心血管疾病、肿瘤疾病的发生和颉颃有毒元素等多种重要的生物学功能。硒以硒半胱胺酸的形式掺入硒蛋白中,通过后者发挥其生物学功能。益生菌具有改善动物胃肠道微生态菌群,抑制胃肠道致病菌的入侵,调节免疫功能,提高饲料利用率和促进生长等生物学功能。前人的研究表明,与无机硒(如亚硒酸钠)相比,有机硒(如富硒酵母,其主要含硒蛋氨酸)的毒性小,而且生物利用率高。富硒益生菌是本实验室研制的饲料添加剂产品,能同时发挥有机硒和益生菌的双重生物学功能,已获国家发明专利。本文研究了日粮添加富硒益生菌对奶牛硒吸收转化、抗氧化能力、泌乳性能和乳中体细胞数的影响,并在细胞和分子水平上探讨其作用机理,为富硒益生菌在奶牛生产上的广泛应用提供理论依据。试验1.日粮添加富硒益生菌对奶牛硒吸收转化效果的影响在考虑试验奶牛的预产期、胎次、上一年度产奶量、年龄和体况分相近的基础上,把36头经产荷斯坦奶牛随机分成4个试验处理组。对照组喂给基础日粮；富硒益生菌(Se-Pro)组喂给基础日粮+Se-Pro 108.2 g/头·天(相当于每头每天补给5 mg Se和320.27x107个活菌数)；益生菌(Pro)组喂给基础日粮+Pro 20.8 g/头·天(相当于每头每天补给320.32x107个活菌数)；亚硒酸钠(Na2SeO3)组喂给基础日粮+Na2SeO3溶液5 ml/头·天(相当于每头每天补给5 mg Se)。试验期为产前30天至产后3个月。于试验的第0、30、60、90和120 d,采集血液样品,用于分析全血Se含量。于产后的第2、30、60和90 d,采集牛奶样品,用于分析乳Se含量。结果,在添饲后的第30、60、90和120 d, Se-Pro组和Na2SeO3组的全血Se含量均显著高于对照组和Pro组(P<0.05)；在产后的第2、30、60和90 d, Se-Pro组和Na2SeO3组的初乳和常乳Se含量均显著高于对照组和Pro组(P<0.05); Se-Pro组的全血和常乳Se含量均高于Na2SeO3组；Pro组与对照组的全血、初乳和常乳Se含量差异均不显著(P>0.05)。结果表明,富硒益生菌提高全血、初乳和常乳Se含量的效果优于亚硒酸钠。试验2.日粮添加富硒益生菌对奶牛机体抗氧化能力的影响试验设计同试验1。试验期为产前30天至产后3个月。于试验的第0、30、60、90和120 d,采集血液样品,用于分析红细胞GPx1活性、血清GPx3活性和血浆MDA含量。结果,在添饲后的第90和120 d, Se-Pro组和Na2SeO3组的红细胞GPx1活性均显著高于对照组和Pro组(P<0.05),而Pro组与对照组的红细胞GPx1活性差异均不显著(P>0.05)；在整个试验期间,Se-Pro组、Pro组和Na2SeO3组的血清GPx3活性与对照组相比差异均不显著(P>0.05)；在添饲后的第30、60、90和120 d, Se-Pro组和Na2SeO3组的血浆MDA含量均显著低于对照组和Pro组(P<0.05),且Se-Pro组的血浆MDA含量均显著低于Na2SeO3组(P<0.05)。结果表明,在提高奶牛机体抗氧化能力和降低血浆MDA含量方面,富硒益生菌优越于亚硒酸钠。试验3.日粮添加富硒益生菌对奶牛泌乳性能和乳中体细胞数的影响试验设计同试验1。试验期为产前30天至产后3个月。产后每个月测产奶量。于产后的第30、60和90 d,采集牛奶样品,用于分析乳成分(乳脂、乳蛋白和乳糖)和体细胞。结果,泌乳期前3个月平均日产奶量(换算成4%乳脂校正奶),Se-Pro组高出对照组2.96 kg·d-1, Pro组高出对照组1.64 kg·d-1, Na2SeO3组高出对照组0.69 kg·d-1。日粮添加富硒益生菌、益生菌或亚硒酸钠对牛奶的乳脂率和乳蛋白率没有影响。Se-Pro组的乳糖率显著高于对照组和Na2SeO3组(P<0.05),而与Pro组比差异不显著(P>0.05)。Se-Pro组的平均牛奶体细胞(SCC)显著低于对照组(P<0.05),但与Na2SeO3组和Pro组差异均不显著(P>0.05)；平均SCC由低到高排序为：Se-Pro组< Na2SeO3组<Pro组<对照组。结果表明,富硒益生菌有提高奶牛产奶量的趋势。富硒益生菌对乳脂率和乳蛋白率没有影响,但能显著提高乳糖率；富硒益生菌降低牛奶SCC的效果比益生菌和亚硒酸钠好。试验4.原代牛肝细胞的分离和培养方法的建立本试验采用胶原酶灌注消化法,对来自新生小牛血清制备厂的牛肝脏尾状突材料进行肝细胞分离,用台盼蓝拒染法测定总细胞数和肝细胞即时存活率,以每孔1×106个肝细胞的量接种于牛尾胶原包被的6孔细胞培养板,进行单层培养细胞形态学观察和培养基上清液LDH活力测定。结果,每头小牛肝脏尾状突分离获得的细胞产量为(3.558±0.228)×108个,肝细胞即时存活率为(84.46±3.56)%,培养24-72 h阶段的肝细胞生长状态最好,适合用于有关肝细胞代谢、中毒、基因表达的研究。试验5.牛肝细胞中GPx1、GPx4及D1mRNA表达水平Real-time PCR检测方法的建立根据已知牛的β-actin、GPx1、GPx4和D1基因的mRNA序列,应用生物软件辅助设计并合成4对特异性引物；以Oligo dT为引物,对从鸡肝细胞中抽提的总RNA进行反转录,合成第一链cDNA;以cDNA为模板,分别进行4个目的基因片段的PCR扩增；胶回收PCR产物,把它们克隆到pMD18-T载体上并测序；对抽提的重组质粒进行拷贝数测定,以10倍梯度稀释的质粒作为标准模板进行PCR扩增,同时对荧光定量PCR反应的引物浓度和退火温度进行优化。结果,4对引物分别扩增出一条目的带,且大小与预期相符合；荧光定量PCR反应的最佳引物浓度是10μmol·L-1,最佳退火温度为62℃；扩增β-actin、GPx1、GPx4和D1基因的4对引物的扩增效率分别为91.99%、96.04%、96.51%和91.61%；各荧光定量PCR产物熔解曲线均显示单一峰。结果表明,本试验成功地建立了检测牛肝细胞中GPx1、GPx4和D1mRNA水平的荧光定量PCR方法。试验6.不同硒形式和浓度对原代培养牛肝细胞GPxl表达的调控来自新生小牛(1-2日龄)的原代肝细胞单层用0(对照)、0.5、1、1.5、2、3、4和5μmol·L-1(以Se计)硒蛋氨酸(Se-Met)或亚硒酸钠(Na2SeO3)或硒卡拉胶(Se-Car)孵育培养24 h。结果,0.5-5μmol·L-1 Se-Met、0.5-1μmol·L-1 Na2SeO3和0.5μmol·L-1 Se-Car处理组的LDH漏出显著低于对照组,但是2-5μmol·L-1 Na2SeO3和3-5μmol·L-1 Se-Car处理组的LDH漏出显著高于对照组,呈剂量依赖模式；所有Se处理组的细胞内还原型GSH浓度均显著低于对照组；所有Se处理组的GPx1 mRNA水平均显著提高,最大值分别出现在3μmol·L-1 Se-Met、1.5μmol·L-1 Na2SeO3和2μmol·L-1 Se-Car;而且3μmol·L-1 Se-Met处理组的GPx1 mRNA水平在所有Se处理组中最高；在达到最高水平之后,再增加Se浓度则导致GPx1 mRNA水平下降；除了变化幅度小些外,GPx1活性的变化模式与GPx1 mRNA的相似。结果表明,不同Se形式和浓度对原代培养牛细胞GPx1 mRNA水平和活性的调控是有差异的；支持牛肝细胞GPx1充分表达的不同Se形式的最佳浓度各有不同；Se-Met的作用效果最好。试验7.不同硒形式和浓度对原代培养牛肝细胞GPx4和D1mRNA表达的影响来自新生小牛(1-2日龄)的原代肝细胞单层用0(对照)、0.5、1、1.5、2、3、4和5μmol·L-1(以Se计)硒蛋氨酸(Se-Met)或亚硒酸钠(Na2SeO3)或硒卡拉胶(Se-Car)孵育培养24 h。结果,与对照组比,所有Se处理组的牛肝细胞GPx4 mRNA水平均显著降低,而所有Se处理组的牛肝细胞D1 mRNA水平均显著升高；不同Se形式处理组的D1mRNA最大值分别出现在1.5μmol·L-1 Se-Met组、1μmol·L-1 Na2SeO3组和1μmol·L-1 Se-Car组；而且1.5μmol·L-1 Se-Met处理组的D1 mRNA水平在所有Se处理组中最高；在达到最大值后,再增加Se-Met、Na2SeO3或Se-Car浓度则导致D1 mRNA水平下降,且呈剂量依赖模式。结果表明,满足牛肝细胞GPx4 mRNA充分表达的Se浓度相对较低(推测<0.5μmol/L);不同Se形式在调控牛肝细胞D1 mRNA表达方面存在差异；支持牛肝细胞D1 mRNA充分表达的不同Se形式的浓度各有不同；Se-Met的作用效果最好。更多还原

【Abstract】 Selenium (Se) is an essential trace element for animals and humans. Se has many biological functions including enhancing the antioxidative ability, immune function, reproductive performance, and reducing the risk of diabetes, cardiovascular disease, certain types of cancer and resisting to detoxification from some heavy metals. Se is incorporated into selenoproteins as selenocysteine and exerts its biological functions. Expression and activity of selenoenzymes are regulated by Se. Probiotics possess many beneficial effects including improving gastrointestinal flora, resisting to infection of enteropathogenic bacteria, regulating immune function, increasing availability of diets and promoting growth. Precious studies have suggested that organic forms of Se (e.g., Se-enriched yeast, which contains selenomethionine) are less toxic than inorganic forms of Se such as sodium selenite, and the bioavailability of organic Se is relatively high. The preparation of Se-enriched Probiotics was made in our lab and used for nutritional supplementation in diets, which can exert dual effects of organic Se and probiotics at the same time. Invention patent for the preparation was awarded by State Intellectual Property Office of The P.R.C. The present study was conducted to evaluate the effects of supplementary Se-enriched probiotics on transform of Se, antioxidative ability, milk production and somatic cell count (SCC) in dairy cattle, and to explore its mechanism at the cellular and molecular levels, so that to provide a theoretical basis for application of Se-enriched probiotics in dairy cattle industry.Experiment 1. Effect of supplementary selenium-enriched probiotics on absorption and transform of selenium in dairy cattleThirty-six multiparous Holstein cows were divided into four groups, as similar as possible with respect to expected calving date, parity, the previous year’s lactation averages, age and body condition score. The control group received a basal diet. The selenium-enriched probiotics (Se-Pro) group received a basal diet plus 108.2 g of Se-Pro/d per cow (mean 5 mg of Se and 320.27x107 cfu/d per cow). The probiotics (Pro) group received a basal diet plus 20.8 g of Pro/d per cow (mean 320.32×107 cfu/d per cow). The sodium selenite (Na2SeO3) group received a basal diet plus 5 ml of Na2SeO3 solution/d per cow (mean 5 mg of Se/d per cow). Experiment period was from d 30 prepartum to 3 mo postpartum. Blood samples were collected for analysis of whole blood selenium (Se) concentrations on d 0,30,60,90 and 120. Milk samples were collected for analysis of milk Se concentrations on d 2,30,60 and 90 of postpartum. Se-Pro group and Na2SeO3 group had significantly higher Se concentrations in whole blood than did control group and Pro group (P<0.05) on d 30,60,90 and 120, respectively. Se-Pro group and Na2SeO3 group had significantly higher Se concentrations in colostrum and milk than did control group and Pro group (P<0.05) on d 2,30,60 and 90 of postpartum, respectively. The Se concentrations of Se-Pro group in whole blood and milk were significantly higher than that of Na2SeO3 group (P<0.05), respectively. The Se concentrations in whole blood, colostrum and milk were not different between Pro group and control group (P>0.05). The results suggest that the effects of Se-Pro are greater than that of Na2SeO3 on increase of Se concentrations in whole blood, colostrum and milk.Experiment 2. Effect of supplementary selenium-enriched probiotics on antioxidative ability of dairy cattleExperiment design is the same as the experiment 1. Experiment period was from d 30 prepartum to 3 mo postpartum. Blood samples were collected for analysis of erythrocyte glutathione peroxidase (RBC GPx1) activity, serum GPx3 activity and plasma Malondialdehyde (MDA) concentrations on d 0,30,60,90, and 120. Se-Pro group and Na2SeO3 group had significantly higher RBC GPx1 activities than did control group and Pro group (P<0.05) on d 90 and 120, respectively, while there were no differences between Pro group and control group (P>0.05). The serum GPx3 activities were not different between the treatment groups and the control group in the whole experiment period (P >0.05). The plasma MDA concentrations in Se-Pro group and Na2SeO3 group were significantly lower than that in control group and Pro group (P<0.05) on d 30,60,90 and 120, respectively. Furthermore, the plasma MDA concentrations in Se-Pro group were significantly lower than that in Na2SeO3 group (P<0.05) on d 30,60,90 and 120, respectively. The results suggest that the effects of Se-Pro are greater than that of Na2SeO3 on increase of antioxidative ability and on reduction of plasma MDA concentrations in dairy cattle. Experiment 3. Effect of supplementary selenium-enriched probiotics on milk production and somatic cell count in dairy cattleExperiment design is the same as the experiment 1. Daily milk yields were determined once monthly after calving. Milk samples were collected for analysis of milk component (fat, protein, and lactose) and somatic cell count (SCC) on d 30,60 and 90 of postpartum. Mean daily milk yield (expressed as 4% fat-corrected milk) for the first 3 mo of lactation was 2.96,1.64 and 0.69 kg-d-’higher in Se-Pro group, Pro group and Na2Se03 group than in control group, respectively. Milk fat percentage and protein percentage were not affected by supplementation of Se-Pro, Pro or Na2SeO3. Milk lactose percentage in Se-Pro group was significantly higher than that in Na2SeO3 group and control group (P< 0.05), but not different when compared with Pro group (P> 0.05). Mean SCC in milk in Se-Pro group was significantly lower than that in control group (P< 0.05), but not different when compared with Na2Se03 group and Pro group (P> 0.05), respectively. Mean SCC from low to high ranking was Se-Pro group< Na2SeO3 group< Pro group< control group. The results suggest that (i) there is a trend for Se-Pro to increase milk yield, (ii) milk fat percentage and protein percentage are not affected by Se-Pro supplementation, whereas milk lactose percentage is significantly increased by Se-Pro supplementation and (iii) the effects of Se-Pro are greater than that of Na2SeO3 and Pro on reduction of milk SCC.Experiment 4. Development of the method for isolation and primary culture of bovine hepatocytesIn the present study, collagenase perfusion method was used to isolate the bovine hepatocytes of caudate lobe obtained from a neonatal calf serum factory. Total cell count and survival rate of freshly isolated hepatocytes were determined by trypan blue exclusion. Hepatocytes were seeded onto a cattle tail collagen coated 6-well microplate at a density of 1×106cells/well. Morphological assessment of hepatocyte monolayers and determination of LDH activity in the culture supernatant were performed. The results showed that the cell yield was 3.558±0.228×108 hepatocytes per caudate lobe. The survival rate of freshly isolated hepatocytes was 84.46%±3.56%. The optimal growth state of hepatocyte monolayers was observed during the 24-72 h culture period, which was applicable in studies relating to hepatocytes metabolism, toxicosis, and gene expression.Experiment 5. Development of the method for determination of GPx1, GPx4, and D1 mRNA levels in bovine hepatocytes by real-time PCRAccording to the published mRNA sequences of gene ofβ-actin, GPxl, GPx4, and D1, four pairs of specific primers were designed by biological software and synthesized. Oligo dTs were used as primer and first strand cDNAs were synthesised from total RNA, which was isolated from bovine hepatocytes. Using the synthesised cDNAs as template, four target fragments were amplified by polymerase chain reaction (PCR), respectively. The PCR products were electrophoresed on 2% agarose gels and extracted. The extracted PCR products were cloned into pMD18-T Vector and sequenced. The copies of extracted plasmids were measured and serially diluted by 10-folds. The diluted plasmids were used as standard templates and real-time PCRs were performed. In addition, the contents and annealing temperatures of four pairs of specific primers were optimized, respectively. The results showed that one target band was amplified for each specific primer pair and consistent with the fact. The optimal contents and annealing temperatures of four primer pairs were 10μmol·L-1 and 62℃, respectively. Amplification efficiencies of four primer pairs for gene ofβ-actin, GPx1, GPx4, and D1 were 91.99%,96.04%,96.51%, and 91.61%, respectively. The melting curve analysis showed only one peak for each PCR product. These results indicate that the method for determination GPx1, GPx4 and D1 mRNA levels in bovine hepatocytes by real-time PCR is successfully developed.Experiment 6. Regulation of cellular glutathione peroxidase by different forms and concentrations of selenium in primary cultured bovine hepatocytesPrimary cultured bovine hepatocyte monolayers derived from neonatal Holstein male calves (aged 1-2 d) were incubated for 24 h with 0 (control),0.5,1,1.5,2,3,4, or 5μmol·L-1 Se as DL-selenomethionine (Se-Met), sodium selenite (Na2SeO3), or Kappa-selenocarrageenan (Se-Car). Compared to controls, significantly lower lactic dehydrogenase (LDH) release was observed at 0.5-5μmol·L-1 Se-Met,0.5-1μmol·L-1 Na2SeO3 and 0.5μmol·L-1 Se-Car, but significantly higher LDH release was observed at 2-5μmol·L-1 Na2SeO3 and 3-5μmol·L-1 Se-Car in a dose-dependent manner. Intracellular reduced glutathione (GSH) contents in all Se-treated hepatocytes were significantly lower than controls. Significant increases of GPxl mRNA level were obtained in all Se-treated hepatocytes, with maximal effects at 3μmol·L-1 Se-Met,1.5μmol·L-1 Na2SeO3 and 2μmol·L-1 Se-Car, respectively. Furthermore,3μmol·L-1 Se from Se-Met resulted in the peak GPx1 mRNA level in all groups treated with Se. After reaching a maximal increase, higher Se supplementation led to reduction of GPxl mRNA level. GPx1 activity showed similar patterns with less magnitude. We conclude that (i) regulation of GPx1 mRNA level and activity by different Se forms are different in primary cultured bovine hepatocytes, (ⅱ) the optimal concentrations of different Se forms are various, which support the full expression of GPx1 in bovine hepatocytes and (ⅲ) the effects of Se-Met are best.Experiment 7. Effects of different forms and concentrations of selenium on mRNA expression of phospholipid hydroperoxide glutathione peroxidase and typeⅠiodothyronine deiodinase in primary cultured bovine hepatocytesPrimary cultured bovine hepatocyte monolayers derived from neonatal Holstein male calves (aged 1-2 d) were incubated with 0 (control),0.5,1,1.5,2,3,4, or 5μmol·L-1 Se as DL-selenomethionine (Se-Met), sodium selenite (Na2SeO3), or Kappa-selenocarrageenan (Se-Car) for 24 h. Compared to controls, significantly lower GPx4 mRNA was observed in all Se-treated hepatocytes, whereas significantly higher D1 mRNA was observed in all Se-treated hepatocytes. The maximal increases of D1 mRNA in the hepatocytes treated with different Se forms were observed at 1.5μmol·L-1 Se-Met,1μmol·L-1 Na2SeO3 and 1μmol·L-1 Se-Car, respectively. Furthermore,1.5μmol·L-1 Se from Se-Met resulted in the peak D1 mRNA level in all Se-treated hepatocytes. After reaching a maximal increase, higher concentration of Se as Se-Met, Na2SeO3, or Se-Car led to reduction of D1 mRNA in a dose-dependent manner. The data suggest that (ⅰ) Se concentration required for full expression of GPx4 mRNA in bovine hepatocytes is relatively low (speculated<0.5μmol/L), (ⅱ) regulation of D1 mRNA by different Se forms is different in bovine hepatocytes, (ⅲ) the optimal concentrations of different Se forms are various, which support the full expression of D1 in bovine hepatocytes and (iiii) the effects of Se-Met are best.更多还原