芦丁对奶牛泌乳性能、瘤胃消化代谢和对大鼠乳腺发育的影响
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摘要
芦丁是一种从植物中提取的黄酮类物质,广泛存在于自然界,现有研究证明,对于人类来说芦丁既可以作为治疗药物,又能够作为保健品使用。奶牛生产性能的发挥从消化生理和泌乳生理角度分析,在很大程度上依赖于瘤胃内营养物质的消化代谢和泌乳器官乳腺的发育。本研究以泌乳奶牛和Wistar大鼠为研究对象和试验动物,就芦丁对奶牛泌乳性能、瘤胃内环境和消化代谢以及动物乳腺发育及泌乳维持的作用展开试验研究,从营养学和生理学方面探索芦丁对奶牛生理营养作用及其调控乳腺发育的作用机制和使用效果。具体研究内容分为以下十个部分:
试验一、芦丁对奶牛泌乳性能的影响
研究芦丁对奶牛泌乳性能的影响,探索芦丁在日粮中的适宜添加剂量,为芦丁在奶牛营养与饲料中的科学使用提供依据。选用生理阶段相近的经产中国荷斯坦奶牛20头,随机分成4组,每组5头。分别在基础日粮中每日每头添加芦丁0.0(对照组)、1.5、3.0、4.5mg/kg。试验期11周,其中预饲期1周,正试期10周。隔日记录试验牛的产奶量,并于试验第5、35、65日采集乳样进行乳成分分析。结果显示,与对照组相比,3.0mg/kg·BW和4.5mg/kg·BW芦丁处理组都显著提高了奶牛的产奶量(P<0.05),而1.5 mg/kg·BW芦丁处理组与对照组奶牛产奶量差异不显著(P>0.05);1.5 mg/kg·BW芦丁处理和3.0mg/kg·BW处理乳脂率虽低于对照组(P<0.05),但仍在正常值范围内,4.5mg/kg·BW芦丁处理乳脂率同对照组差异不显著(P>0.05),各处理组间牛乳乳蛋白、乳糖、非脂固形物差异不显著(P>0.05)。试验表明:芦丁能够提高奶牛泌乳性能,同时保持主要乳成分不受影响,在本试验条件下,芦丁适宜添加量为3.0mg/kg·BW。
试验二、芦丁在奶牛胃肠道不同部位出现的峰值研究
研究芦丁在奶牛胃肠道不同部位出现的峰值及其特点。利用4头安装有永久性瘤胃瘘和十二指肠瘘的成年奶牛,采用自身配对设计,对照期饲喂基础全混合日粮(TMR),试验期每日每头在基础日粮中一次性投喂芦丁3.0mg/kg·BW,在投喂后1 ~ 8 h每小时采集瘤胃液、十二指肠液和血液,检测其中芦丁和槲皮素的含量。结果显示,芦丁和槲皮素在奶牛瘤胃液中分别于饲喂后1 h和2 h达到峰值3.017±0.153μg/ml和1.148±0.153μg/ml;十二指肠液中在6 h出现峰值2.133±0.057μg/ml和0.214±0.004μg/ml;在血液中芦丁和槲皮素均出现两个峰值,芦丁的峰值出现在1 h(0.180±0.029μg/ml)和8 h(0.229±0.014μg/ml),槲皮素的峰值在1h(0.064±0.002μg/ml)和8h(0.089±0.007μg/ml)。试验表明:芦丁及其代谢产物槲皮素在奶牛胃肠道中出现的峰值因饲喂时间和消化部位不同而有差异。
试验三、芦丁对奶牛血液生化指标和激素水平的影响
在奶牛日粮中添加芦丁,测定奶牛血液生化指标、免疫指标和激素水平的变化,旨在探索芦丁对奶牛血清学指标和泌乳相关激素水平的影响。选用生理状态等相近的经产中国荷斯坦泌乳牛10头,随机分成2组,每组5头。分别在基础日粮中每日每头添加芦丁0.0 mg/kg·BW(对照组)和3.0 mg/kg·BW(试验组)。试验期11周,其中预饲期1周,正试期10周,在试验第5、35、65日采集血样,检测其生化、免疫和激素指标。试验组与对照组相比,总蛋白、白蛋白、葡萄糖水平差异不显著(P>0.05),芦丁可以显著降低奶牛血液中尿素氮的含量(P<0.05);IgG、谷丙转氨酶(ALT)、谷草转氨酶(AST)、碱性磷酸酶(ALP)、皮质醇(Cort)含量两组差异不显著(P>0.05),试验组溶菌酶(LZM)水平显著高于对照组(P<0.05);试验组和对照组奶牛血液中雌激素(E2)、孕激素(P)和催乳素(PRL)差异不显著(P>0.05),试验组催乳素水平由试验第5日12.19 ng/ml至第65日提高到13.94 ng/ml呈现上升趋势。试验显示:芦丁能够降低奶牛血液中尿素氮水平,提高溶菌酶含量。
试验四、芦丁对奶牛瘤胃消化代谢的影响
研究芦丁对奶牛瘤胃消化代谢的影响,旨在为探讨芦丁对奶牛瘤胃发酵的调控作用提供数据与参考。利用安装有永久性瘤胃瘘和十二指肠瘘的4头成年奶牛,采用自身配对设计,对照期饲喂TMR ,试验期每日每头在TMR中一次性投喂芦丁3.0mg.kg·BW,在投喂后1 h ~ 8 h每小时采集瘤胃液,检测瘤胃液中pH值、氨态氮(NH3-N)、挥发性脂肪酸(VFA)、瘤胃原虫数量、微生物蛋白的变化情况。结果显示,试验期瘤胃液pH值5.92低于对照期6.16(P<0.05),NH3-N浓度11.03 mg/100ml低于对照期20.07 mg/100ml(P<0.05),总挥发性脂肪酸(TVFA)试验期96.72 mmol/L高于对照期83.12 mmol/L(P<0.05),乙酸浓度61.00 mmol/L高于对照期51.47 mmol/L(P<0.05),丙酸浓度36.35 mmol/L高于对照期19.95 mmol/L(P<0.05),乙酸/丙酸的比例试验期与对照期差异不显著(P>0.05),乙酸、丙酸在TVFA中所占的比例试验期与对照期差异也不显著,试验期中瘤胃原虫数12.54(×105个/ml)低于对照期16.37(×105个/ml)(P<0.05),原虫蛋白含量试验期3.71 mg/ml低于对照期5.14 mg/m(lP<0.05),菌体蛋白含量试验期1.50 mg/ml高于对照期0.86 mg/ml(P<0.05)。试验表明:芦丁具有以下作用,降低瘤胃中NH3-N浓度,增加挥发性脂肪酸的总量,增加乙酸、丙酸的浓度,降低瘤胃pH值,抑制瘤胃原虫数量,降低原虫蛋白含量,增加瘤胃液中菌体蛋白的浓度。
试验五、体外法研究芦丁对奶牛瘤胃主要消化酶活性的影响
研究芦丁对奶牛瘤胃主要消化酶活性的影响。选择体况良好,体重600 kg左右,安装有永久性瘤胃瘘管的健康中国荷斯坦奶牛5头,采集瘤胃内容物制取固相和液相酶液,分别添加芦丁0.00 mg/ml(对照组)、0.02 mg/ml、0.04 mg/ml、0.06 mg/ml,混匀后检测其中淀粉酶、滤纸酶、β-葡萄糖苷酶、羧甲基纤维素酶、微晶纤维素酶、果胶酶、木聚糖酶的活性。结果显示,添加芦丁的试验组与对照组相比较,奶牛瘤胃内容物固相和液相总脱氢酶、滤纸酶、β-葡萄糖苷酶、羧甲基纤维素酶和微晶纤维素酶的活性均有显著差异(P<0.05),蛋白酶活性显著低于对照组(P<0.05),而淀粉酶、果胶酶与木聚糖酶与对照组相比较其酶活性差异不显著(P>0.05)。试验表明:芦丁能够提高奶牛瘤胃内容物固相和液相中总脱氢酶、滤纸酶、β-葡萄糖苷酶、羧甲基纤维素酶、微晶纤维素酶的活性。
试验六、体外法研究芦丁对牛初乳酶活的影响
利用奶牛分泌到初乳中的生物活性物质——酶作为指标,来分析和评价芦丁对奶牛乳腺细胞的作用。采集5头健康中国荷斯坦奶牛分娩后第二天的初乳,向初乳样品中添加5个芦丁浓度剂量梯度0 mol/L(对照)、10~(-7) mol/L、10~(-6) mol/L、10~(-5) mol/L、10~(-4) mol/L,混匀后检测初乳中β-N-乙酰氨基葡萄糖苷酶(NAG)、碱性磷酸酶(ALP)、乳过氧化物酶(LP)、γ-谷氨酰转肽酶(γ-GT)的活性。检测结果显示10~(-6) mol/L、10~(-5) mol/L、10~(-4) mol/L剂量组NAG酶比活力分别为7.61、6.92、6.43(U/L)显著低于对照组9.74(U/L)(P<0.05),10~(-7) mol/L剂量组NAG酶比活力为8.17 U/L与对照组相比差异不显著(P>0.05);各剂量组ALP酶活性差异不显著;10~(-7) mol/L、10~(-6) mol/L、10~(-5) mol/L、10~(-4) mol/L各剂量组LP酶比活力分别为58.30、65.00、72.94、77.03(U/ml)显著高于对照组48.89(U/ml)(P<0.05);10~(-7) mol/L、10~(-6) mol/L剂量组γ-GT酶比活力分别为11613.49、11371.1(6U/L),显著高于对照组10248.84(U/L)(P<0.05),10~(-5) mol/L、10~(-4) mol/L剂量组γ-GT酶比活力与对照组相比差异不显著(P>0.05)。本试验结果表明:在试验室条件下,芦丁能够提高奶牛初乳中LP和γ-GT酶活性,抑制NAG酶活性,对ALP影响差异不显著。
试验七芦丁对大鼠泌乳性能的影响
通过研究芦丁对大鼠泌乳性能、内分泌激素、乳腺器官指数的影响,考察芦丁调节大鼠乳腺泌乳和维持泌乳的作用。试验选择18只Wistar受孕母鼠,随机分为3组,每组6只,分别为对照组(每日每只灌胃生理盐水2ml)、芦丁组(每日每只灌服芦丁60 mg/kg·BW)、雌二醇组(每周每只肌注雌二醇60ug/kg·BW),从哺乳第4天开始连续给药2周,基础日粮相同。测定指标包括:大鼠泌乳量,仔鼠平均体增重,乳腺器官指数,血浆与乳腺组织中雌激素(E2)、孕激素(P)、催乳素(PRL)、生长激素(GH)的含量。试验结果显示,芦丁组大鼠泌乳量7.35(g/h)显著高于对照组5.73(g/h)(P<0.05),与雌二醇组8.22(g/h)差异不显著(P>0.05);芦丁组与雌二醇组相比,仔鼠体增重差异不显著(P>0.05),但芦丁组与雌二醇组仔鼠体增重显著大于对照组(P<0.05);芦丁组大鼠血浆与乳腺组织中E2、PRL、GH的水平显著高于对照组(P<0.05),低于雌二醇组(P<0.05),P水平各组间差异不显著(P>0.05);雌二醇组和芦丁组大鼠乳腺器官指数显著高于对照组(P<0.05)。试验结果表明:在本试验条件下,芦丁能够促进乳腺分泌乳汁,显著提高雌性大鼠泌乳量,对促进大鼠乳腺器官的发育有一定作用,能发挥类雌二醇样作用。
试验八芦丁对雌性青春期大鼠乳腺发育及相关激素与受体水平的影响
通过研究芦丁对雌性青春期大鼠内分泌激素水平、激素受体表达的影响和乳腺组织形态学变化的观察,探讨芦丁对乳腺发育的作用。选取24只雌性青春期Wistar大鼠,随机分为3组,对照组(Con):每日每只灌胃生理盐水2 ml;芦丁组(Rut):每日每只灌服芦丁60 mg/kg·BW;雌二醇组(Est):每周每只肌注雌二醇60μg/kg·BW。试验期2周,结束时检测大鼠血浆和乳腺组织中雌激素(E2)、孕激素(P)、催乳素(PRL)、生长激素(GH)含量及其受体表达,并进行乳腺组织形态学观察。研究结果显示,Est组、Rut组、Con组大鼠血浆与乳腺组织中E2、PRL水平分别依次降低,差异显著(P<0.05);Rut组P水平显著高于Con组(P<0.05),与Est组相比,差异不显著(P>0.05);Est组GH水平高于Rut组,Rut组GH水平高于Con组,差异均不显著(P>0.05)。雌激素受体(ER)、孕激素受体(PR)、催乳素受体(PRLR)、生长激素受体(GHR)在乳腺组织中的表达量依次为Est组>Rut组>Con组(P<0.05)。Rut组大鼠乳腺组织切片腺泡管腔直径大于Con组(P<0.05),小于Est组(P>0.05)。试验表明:在本试验条件下,芦丁能够提高雌性青春期大鼠E2、P水平,促进PRL分泌,上调乳腺组织中ER、PR、PRLR、GHR的表达量,促进乳腺发育,发挥类雌二醇样作用。
试验九芦丁对去卵巢处女大鼠乳腺发育作用的影响
研究芦丁对去卵巢处女大鼠内分泌激素水平、激素受体表达的影响以及乳腺组织形态变化,旨在了解和认识芦丁促进乳腺发育的类雌激素样作用功效。试验选择32只去卵巢处女Wistar大鼠,随机分为四个处理组,假手术组(Sham),每日每只灌胃2 mL生理盐水;卵巢切除组(Ova),每日每只灌胃2 mL生理盐水;去卵巢芦丁组(Ova+Rut),每日每只按60 mg/kg·BW剂量灌服芦丁;去卵巢雌二醇组(Ova+Est),每周每只肌注雌二醇(60 ug/kg·BW)一次。试验为期两周,试验结束后采集大鼠血液样品、乳腺组织样品进行测定分析。试验结果显示,Ova+Rut组和Ova+Est组大鼠血浆中雌激素(E2)的水平高于Ova组(P<0.05),该两组间差异不显著(P>0.05),Ova+Rut组水平低于Sham组(P<0.05),Ova+Est组与Sham组差异不显著(P>0.05),各处理组间乳腺组织中E2含量呈现相同规律;各处理组血浆与乳腺组织中催乳素(PRL)的含量变化趋势为Ova组0.05),Ova+Est组与Sham组差异不显著(P>0.05);Ova组、Ova+Rut组、Ova+Est组、Sham组各组间大鼠乳腺组织中雌激素受体(ER)、催乳素受体(PRLR)、生长激素受体(GHR)表达量依次增大(P<0.05),呈现相同变化趋势;乳腺组织形态学观察发现,Ova+Rut组、Ova+Est组、Sham组乳腺组织的发育显著好于Ova组(P<0.05)。结果表明:在本试验条件下,芦丁能够提高去卵巢处女大鼠E2水平,促进垂体分泌PRL、GH,上调乳腺组织中ER、PRLR、GHR的表达量。
试验十芦丁对哺乳大鼠乳腺激素受体表达量的影响
本试验目的是研究芦丁对哺乳期大鼠泌乳相关激素受体表达量的影响。试验选择18只Wistar哺乳母鼠,随机分为3组,每组6只,分别为对照组(Lac-Con组)每日每只灌胃生理盐水(2 mL)一次、芦丁组(Lac-Rut组)每日每只灌服芦丁(60 mg/kg·BW)一次、雌二醇组(Lac-Est组)每周每只肌注雌二醇(60μg/kg·BW)一次,从哺乳第4天开始连续给药2周,基础日粮相同。检测哺乳大鼠乳腺组织中雌激素受体(ER)、孕激素受体(PR)、催乳素受体(PRLR)、生长激素受体(GHR)的表达量,并对乳腺组织进行形态学观察。试验显示,ER、PR、PRLR、GHR在乳腺组织中的表达量Lac-Est组>Lac-Rut组>Lac-Con组(P<0.05);乳腺腺泡/管腔直径统计显示Lac-Est组和Lac-Rut组高于Lac-Con组(P<0.05),Lac-Est组与Lac-Rut组差异不显著(P>0.05)。试验结果表明:在本试验条件下,芦丁具有诱导ER、PR、PRLR和GHR表达的作用。
综上所述,本文系统地研究了芦丁对奶牛生产性能的影响,对瘤胃消化代谢的调节作用,及对乳腺和机体免疫功能的影响,探讨了芦丁对乳腺发育和维持泌乳的影响及相关内分泌作用。根据试验结果与分析讨论,可以得出以下主要结论,在本研究条件下:芦丁及其代谢物槲皮素在瘤胃液中出现峰值的时间为饲喂后1 h和2 h,在十二指肠液中出峰时间为6 h,在血液中出双峰时间均为1 h和8 h。日粮中按3.0 mg/kg添加芦丁,可以显著提高奶牛产奶量;日粮中添加芦丁可以改善瘤胃发酵和氮代谢,促进氮的代谢和利用,在一定程度上可抑制瘤胃原虫数量,降低原虫蛋白含量,增加瘤胃液中菌体蛋白的浓度。日粮中添加芦丁能够提高奶牛瘤胃内容物固相和液相中总脱氢酶、滤纸酶、β-葡萄糖苷酶、羧甲基纤维素酶、微晶纤维素酶活性,对奶牛免疫功能和激素水平无调节作用,可在一定程度上提高血清中溶菌酶的浓度,降低血液中尿素氮水平。芦丁能够提高奶牛初乳中LP和γ-GT酶活性,抑制NAG酶活性,对ALP影响差异不显著。在Wistar大鼠试验中,芦丁能够促进哺乳大鼠乳腺分泌乳汁,对大鼠乳腺器官的发育有一定的促进作用,能发挥类雌二醇样作用;能够提高雌性青春期大鼠E2、P水平,促进PRL分泌,上调乳腺组织中ER、PR、PRLR、GHR的表达量,促进乳腺发育,有类雌二醇样作用的功效;能够提高去卵巢处女大鼠E2水平,促进垂体分泌PRL、GH,上调乳腺组织中ER、PRLR、GHR的表达量;具有诱导ER、PR、PRLR和GHR表达的作用。
Rutin is the flavonoids extracted from the plant, and is widely distributed in nature. Studies have shown that rutin can be used not only in treatments but also as health care products for humans. From perspectives of digestive physiology and the physiology of lactation, the performance of dairy cows depends to a large extent on the rumen digestion and metabolism of nutrients and the development of lactating mammary gland.
In this series of researches, dairy cows and Wistar rats were used as animal models to study the effects of rutin supplementation on the production performance of diary cows, internal environment, digestion and metabolism of rumen, and the development and lactation maintenance of mammary gland. Besides, the action mechanisms and using effects of rutin on dairy cows physiological nutrition and mammary gland development were explored from aspects of nutriology and physiology. The present research consists of ten experiments which were listed as follows:
EXP 1. Effect of Rutin Supplementation on Lactation Performance
This study was designed to investigate the effects of rutin supplementation on milk yield in dairy cows, to find the optimum dosage of rutin and subsequently to offer the scientific basis for the usage of rutin in cows nutrition and feed. Twenty cows in their peak lactation were randomly divided into 4 groups and each was offered a basal diet supplemented with 0, 1.5, 3.0 or 4.5 mg rutin/kg·BW of diet. The trial lasted 11 weeks, including an adaption period of one week and a ten-week experimental period. Milk yields were recorded every other day, and milk compositions were analyzed on the 5th, 35th and 65th day. The experiment showed that, compared to the control group, the cows receiving 3.0 and 4.5 mg·rutin/kg·BW had significantly higher milk yield (P<0.05). No significant difference in milk yield was detected between the cows receiving 1.5 mg·rutin/kg·BW and the control group (P > 0.05). The milk fat content of the cows receiving 1.5 or 3.0 mg·rutin/kg·BW was lower than that of the control group (P<0.05). No significant difference in the milk fat content was observed between the cows offered 4.5 mg·rutin/kg·BW and the control group (P>0.05). The contents of milk protein, lactose and milk non-fat solid matter did not differ significantly among groups (P>0.05). Experiments indicated that dietary rutin improved the production performance of dairy cows,while the main milk compositions were not affected. Under this experimental conditions, the appropriate dosage for rutin supplementation was 3.0 mg/kg·BW diet.
EXP 2. Study on the Peak of Rutin in Different Parts of the Gastrointestinal Tract in Dairy Cows
4 adult cows with permanent rumen fistula and duodenal cannulae were attributed to a self control design and used to investigate the time and characteristics of peak occurrence in different parts of the gastrointestinal tract in dairy cows. Total mixed rations ( TMR ) were used as a basal diet in control period. Cows were fed with rutin one at a time per day in test period. The levels of rutin and quercetin in rumen fluid, duodenal content and blood were detected once per hour from 1 h to 8 h after feeding. The results showed that the concentration of rutin and quercetin in rumen fluid reached their peak levels at 1 h ( 3.017±0.153μg/ml ) and 2 h ( 1.148±0.153μg/ml ) respectively. The peak level in duodenal content occurred at 6h ( 2.133±0.057μg/ml and 0.214±0.004μg/ml ). Rutin had two peaks in blood at 1 h ( 0.180±0.029μg/ml )and 8 h(0.229±0.014μg/ml)respectively. So did quercetin at 1h(0.064±0.002μg/ml )and 8h(0.089±0.007μg/ml). These results indicated that feeding time and digest positions affected the peak occurrence of rutin and aglycone quercetin in the gastrointestinal tract in dairy cows.
EXP 3. Effects of Rutin Supplementation on Blood Metabolites and Levels of Hormone in Dairy Cows
By investigating the changes of blood metabolites and levels of hormone in dairy cows after rutin supplementation, we try to study the effects of rutin on immune function and milk-related hormones. Ten cows in their peak lactation were randomly divided into 2 groups and each was offered a basal diet supplemented with 0.0 and 3.0 mg rutin/kg·BW of diet. The trial was 11 weeks, including an adaption period of one week and a ten-week experimental period. The vein blood samples were analyzed on the 5th, 35th and 65th day. Test serum biochemical indexes, immune indexes and lactation-related hormones. Compared to the control group, no significant difference was observed in the serum concentration of TP, ALB and Cort (P>0.05). But rutin could reduce the content of urea nitrogen in blood (P<0.05).There was no significant difference in the serum concentration of IgG, ALT, AST, ALP and cortisol between the cows receiving 3.0 mg·rutin/kg·BW and the control group (P<0.05). The lysozyme content of the experimental group was higher than that of control group (P<0.05). The serum concentration of estrogen (E2) and progestin (P) did not differ significantly between the treatments (P>0.05), but the serum progesterone level which increased from 12.19 ng/ml on the 5th day to 13.94 ng/ml on the 65th day showed an upward trend in rutin-supplemented cows compared to control. It suggested that rutin could decrease blood urea nitrogen and increase serum lysozyme content.
EXP 4. Effects of Rutin on the Ruminal Digestibility and Metabolism of Dairy Cows
The study was designed to investigate the role of rutin on the ruminal digestibility and metabolism of dairy cows, and subsequently to provide data and references to exploring the effect of rutin on ruminal fermentation. 4 adult cows with permanent rumen fistula and duodenal cannulae were attributed to a self control design and used in the investigation. TMR were used as basal diet in the control period. Cows were fed with rutin one at a time per day in the test period. The levels of pH, ammonia nitrogen ( NH3-N ), volatility fatty acid ( VFA ), the number of rumen protozoa and microbial protein ( MCP ) were detected once per hour from 1 h to 8 h after feeding. The results showed that the rumen fluid pH in the test period ( 5.92 ) was lower than that in the control period ( 6.16 ) (P<0.05). The concentration of NH3-N in the test period ( 11.03 mg/100ml ) was less than that in the control period ( 20.07 mg/100ml ) (P<0.05).Compared to the control period( 83.12 mmol/L ) , the concentration of TVFA in the test period ( 96.72 mmol/L ) was higher (P<0.05). Acetic acid in the test period ( 61.00 mmol/L ) was on a higher level compared to the control period ( 51.47 mmol/L ) (P<0.05) and so was propionic acid in the test period ( 36.35 mmol/L ) compared to the control period ( 19.95 mmol/L ) (P<0.05). There were no significant difference in both the proportion of acetic acid and propionic acid in TVFA (P<0.05). The number and protein content of rumen protozoa in the test period ( 12.54×10~5个/ml, 3.71 mg/ml ) were less than those in the control period ( 16.37×10~5个/ml , 5.14 mg/ml ) (P<0.05). In the test period the MCP ( 1.50 mg/ml ) was higher compared to the control period (0.86 mg/ml ) (P<0.05). Those results indicated that rutin could reduce the concentration of NH3-N , increase the TVFA and the concentration of acetic acid and propionic acid, lower pH level, bring down the number and the protein content of rumen protozoa, and increase MCP in the rumen.
EXP 5. Study the Effect of Rutin on Activities of Digestive Enzymes in the Rumen in Vitro
This study was conducted to investigate the effect of rutin on activities of digestive enzymes in the rumen of dairy cows. 5 healthy about 600 kg-weight China Holstein Cows with permanent rumen fistula were used in this study. Rutin was added into the enzyme ( solid and liquid fraction) extracted from rumen content on the dosage of 0.00 mg/ml (control group), 0.02 mg/ml, 0.04 mg/ml, 0.06 mg/ml respectively and. After mixing, the enzyme activities of protease, amylase, total dehydrogenase, filter paper enzyme,β-glucosidase, carboxymethyl cellulose (CMC), avicelase, pectinase, xylanase were measured. The results showed that compared to the control group, the enzyme activities total dehydrogenase, filter paper enzyme,β-glucosidase, CMC, avicelase in experimental groups significantly differed in both liquid and solid fraction ( P<0.05 ). The enzyme activities of protease were lower than control group ( P<0.05 ). There were no significant difference in the activities about amylase, pectinase and xylanase( P>0.05). In all groups, the enzyme activities of solid fraction were significantly higher than that of liquid faction. It suggested that rutin could enhance the enzyme activities of filter paper enzyme,β-glucosidase, CMC, avicelase in solid and liquid fraction in the rumen of dairy cows. The value of the enzyme activities in solid fraction was higher than that in liquid fraction.
EXP 6. Study the Effect of Rutin on the Enzyme Activities of Bovine Colostrum in Vitro
The enzyme ( the bioactive substances ) secreted into colostrum by dairy cows was used as an indicator to analyze and evaluate the action of rutin on cow mammary cells. The colostrum samples of five healthy Chinese Holstein cows was collected the day after delivery. Rutin was added into the samples on the dosage of 0(control)、10~(-7)、10~(-6)、10~(-5)、10-(4mol/L ). After mixing, the enzyme activities ofβ-N-acetylglucosaminidase ( NAG ), alkaline phosphatase ( ALP ), lactoperoxidase ( LP ),γ-glutamyl transpeptidase (γ-GT ) were measured. Results showed that the enzyme activites of NAG in 10~(-6)、10~(-5)、10~(-4)(mol/L )groups ( 7.61、6.92、6.43 U/L respectively)were lower than that in the control group 9.74(U/L)(P<0.05). No significant difference was observed between 10~(-7)(mol/L)group (8.17 U/L) and the control group (P>0.05). The enzyme activities of ALP did not differ significantly among the treatments (P>0.05). The enzyme activities of LP in 10~(-7)、10~(-6)、10~(-5)、10~(-4)(mol/L)groups ( 58.30、65.00、72.94、77.03 U/ml respectively) were higher than the control group ( 48.89 U/ml )(P<0.05). The enzyme activities ofγ-GT in 10~(-7)、10~(-6)(mol/L)groups (11613.49、11371.16U/L respectively)were higher than 0(mol/L)(10248.84U/L)(P<0.05), but there were no significant differences in 10~(-5)、10~(-4)(mol/L)groups 10036.74、9542.33U/L) ompared to the control group(P<0.05). This result suggested that, under such experimental conditions, rutin could improve the enzyme activities of LP andγ-GT, inhibit the enzyme activities of NAG, and exert no significant effect on the enzyme activities of ALP.
EXP 7. Effect of dietary rutin on laction performance in rats
The objective of this study was to investigate the regulatory effect of rutin on lactation of rats by examining the effects of rutin on milk yields, endocrine hormones and mammary organ indexes of rats. Eighteen pregnant Wistar rats were randomly assigned to 3 treatments: control, gastric infusion of rutin at 60 mg/kg·BW per day, and intramuscular injection of estradiol 60μg/kg·BW weekly from the fourth lactation day for two weeks with the same basal diet. The milk yield of rats, body weight gain of the neonatal rats, mammary gland index, the level of estrogen (E2), progesterone (P), prolactin (PRL) and growth hormone (GH) in plasma and gland tissues were measured. The results shown that the milk yield of the rats receiving rutin ( 7.35 g/h ) was higher than that of the control group ( 5.73 g/h ) (P<0.05), but no obvious difference was observed when compared to the estradiol group ( 8.22 g/h ) (P>0.05). The body weight gain of the neonatal rats in the rutin group had no distinct difference with that in the estradiol group (P<0.05), but these two groups were higher than the control group (P>0.05). The level of E2, PRL and GH in plasma and gland tissues of the rats in the rutin group was higher than that in the control group (P<0.05), but less than that in the estradiol group (P<0.05), and the differences of P level among groups were not distinct (P>0.05). The indexes of mammary gland of the rats in the estradiol and rutin group were higher than that in the control group (P<0.05). Those results indicated that, under such experimental conditions, dietary rutin could promote mammary gland to secrete milk , significantly improve the milk yield of female rats, affect to some extent the development of the thymus, and excert E2-like effects.
EXP 8. Effects of Rutin on the Development of Mammary Gland and Levels of Relevant Hormones and Hormonal Receptors of Pubertal Female Rats
The objective of this study was to investigate the regulatory effect of rutin on mammary glands of unmarried rats by studying the plasma level of endocrine hormones, the expression of hormonal receptors and the changes in the histomorphology of mammary tissues. Twenty four intact virgin rats were randomly assigned to three treatments: 1) gastric infusion of 2 ml normal saline per mouse per day (control (Con)); 2) gastric infusion of 60 mg rutin/kg·BW per day (Rutin (Rut)); and 3) intramuscular injection of 60μg estradiol/kg·BW weekly (Estradiol (Est)). The trial lasted two weeks. The concentration of estrogen (E2), progesterone (P), prolactin (PRL) and growth hormone (GH) in plasma and gland tissues was determined, and the expression of estrogen receptors (ER), progesterone receptors (PR), prolactin receptors (PRLR) and growth hormone receptors (GHR) in gland tissues was measured. The histomorphology of mammary tissues was also monitored. Results showed that the concentration of E2 and PRL in plasma and mammary tissues was ranked as Est>Rut>Con, and the differences were distinct (P<0.05), the concentration of P in plasma and mammary tissues in Rut group was higher than that in Con group(P<0.05), and there was no difference between Rut and Est groups(P>0.05).The concentration of GH in Est grouo was higher than that in Rut group, so was Rut group compared to Con group, and the difference was not significant(P>0.05). The expression of ER, PR, PRLR and GHR in mammary tissues was ranked as Est>Rut>Con (P<0.05). The diameter of mammary acinar cells and lumen was higher (P<0.05) in Rut group than that in Con group, but lower than that in Est group (P>0.05). These results suggested that, under such experimental conditions, rutin could improve the plasma concentration of E2 and P in pubertal female rats, promote PRL release, and up-regulate the expression of ER, PR, PRLE and GHR, exert E_2-like effects.
EXP 9. Experiment Study of Rutin on Mammary Gland Development in Ovariectomized Virgin Rats
The objective of this study was to realize the estrogen-like effect of rutin on promoting mammary gland development by examining the levels of endocrine hormones , the expression of hormonal receptor and the changes in the histomorphology of mammary tissues of ovariectomized virgin rats. Thirty-two ovariectomized virgin rats were randomly assigned to four treatments: sham, gastric infusion of 2 ml normal saline per mouse and per day; Ova, gastric infusion of 2 ml normal saline per mouse and per day; Ova+Rut, gastric infusion of 60 mg rutin/kg·BW per day; Ova+Est, intramuscular injection of 60μg estradiol/kg·BW weekly. The trial lasted two weeks. The samples of blood, gland tissues, thymuses and spleens were collected for quantitative analysis. The level of estrogen (E2), prolactin (PRL) and growth hormone (GH) in plasma and gland tissues were detected and the expression of estrogen receptor (ER), prolactin receptor (PRLR) and growth hormone receptor (GHR) in gland tissues were detected. Histomorphology of development about mammary gland were observed. The results showed that, the level of E2 in plasma in Ova+Rut and Ova+Est were higher than that in Ova (P<0.05),and the difference between these two groups was not significant (P> 0.05), Ova+Rut was lower than Sham (P<0.05), Ova+Est and Sham had no distinct difference(P>0.05). The level of E2 in gland tissues showed the same trend among different treatment groups. The order of the PRL in plasma and gland tissues was Ova0.05), the difference between Ova+Est and Sham was not significant either (P>0.05). The expression of ER, PRLR, GHR in gland tissues in Ova, Ova+Rut, Ova+Est and Sham increased in turn (P<0.05), and showed the same trend. The observation of histomorphology showed that the development of gland tissue in Ova+Rut, Ova+Est and Sham was better than that in Ova (P<0.05). The results suggested that under the experimental conditions, rutin could increase the level of E2 in ovariectomized virgin rats, promote pituitary PRL and GH release, up-regulated the expression level of ER, PRLE and GHR.
EXP 10. Effect of Dietary Rutin on the Expression of Hormonal Receptor in Lactating Rats
The objective of this study was to observe effects of rutin on mammary gland development, endocrine hormone level and hormonal receptor expression in lactation rats. Eighteen Wistar rats were randomly assigned to three treatments: control (Lac-Con), gastric infusion of 2 ml normal saline per mouse and per day. Rutin (Lac-Rut), gastric infusion of 60 mg rutin/kg·BW per day. Estradiol (Lac-Est), intramuscular injection of 60μg estradiol/kg·BW weekly. The experiment lasted for two weeks from the fourth lactation day with the same basal diet. The expression of estrogen receptor (ER), progesterone receptor (PR), prolactin receptor (PRLR) and growth hormone receptor (GHR) in gland tissues were measured. Histomorphology of mammary gland development were observed. The results showed that the expression of ER, PR, PRLR and GHR in gland tissues was Lac-Est > Lac-Rut > Lac-Con (P<0.05). The diameter of mammary gland alveolus showed that Lac-Est and Lac-Rut were higher than Lac-Con (P<0.05), there was no significant difference between Lac-Est and Lac-Rut (P>0.05). The results indicated that, under such experimental conditions, rutin had the function of inducing the expression of ER, PR, PRLE and GHR.
To sum up, in this dissertation, we systematically studied the effect of rutin on production performance of dairy cows, the regulating action of rutin on rumen digestion and metabolism, and the influence of rutin on the immune function of the mammary gland and the organism. We also explored the role of rutin on mammary gland development and the maintenance of lactating as well as the mechanisms of related endocrine system. The following main conclusions could be drawn according to the experimental results, analysis and discussions. Under the study conditions rutin had the functions as follows:
The peak time of rutin and its metabolite– quercetin in the rumen fluid occurred at 1h and 2 h after feeding respectively, in the duodenal content the peak time both occurred at 6 h, and in the blood there were two peaks which both occurred at 1h and 8 h. Dietary rutin added on the dosage of 3.0 mg/kg·BW could obviously promote the milk yield and no significant difference was observed in the milk composition. Dietary rutin could improve rumen fermentation and the nitrogen metabolism, promote the nitrogen metabolism and utilization, bring down the number and the protein content of rumen protozoa to some extent, and increase MCP in the rumen. Dietary rutin could enhance the enzyme activities of total dehydrogenase, filter paper enzyme,β-glucosidase, CMC, avicelase in solid and liquid fraction in the rumen of dairy cows, but had no regulatory role on the immune function and hormone level. It could also increase serum lysozyme content to some degree, and decrease blood urea nitrogen level. Rutin could improve the enzyme activities of LP andγ-GT, inhibit the enzyme activities of NAG, and exert no significant effect on the enzyme activities of ALP in the colostrum of dairy cows. In the experiments using rats as animal model, rutin could promote mammary glands of pregnant rats to secrete milk, have some effect on the development of the rat’s thymus, and excert E2-like effects. Rutin could also increase the level of E2 and P in pubertal female rats, promote PRL release, and up-regulate the expression of ER, PR, PRLE and GHR, promote the development of mammary glands, and exert E2-like effects; Rutin could increase the level of E2 in ovariectomized virgin rats, promote pituitary PRL and GH release, up-regulated the expression level of ER, PRLE and GHR; It could also increase the level of E2 in ovariectomized virgin rats, promote pituitary PRL and GH release, up-regulated the expression level of ER, PRLE and GHR, and have the function of inducing the expression of ER, PR, PRLE and GHR.
试验一、芦丁对奶牛泌乳性能的影响
研究芦丁对奶牛泌乳性能的影响,探索芦丁在日粮中的适宜添加剂量,为芦丁在奶牛营养与饲料中的科学使用提供依据。选用生理阶段相近的经产中国荷斯坦奶牛20头,随机分成4组,每组5头。分别在基础日粮中每日每头添加芦丁0.0(对照组)、1.5、3.0、4.5mg/kg。试验期11周,其中预饲期1周,正试期10周。隔日记录试验牛的产奶量,并于试验第5、35、65日采集乳样进行乳成分分析。结果显示,与对照组相比,3.0mg/kg·BW和4.5mg/kg·BW芦丁处理组都显著提高了奶牛的产奶量(P<0.05),而1.5 mg/kg·BW芦丁处理组与对照组奶牛产奶量差异不显著(P>0.05);1.5 mg/kg·BW芦丁处理和3.0mg/kg·BW处理乳脂率虽低于对照组(P<0.05),但仍在正常值范围内,4.5mg/kg·BW芦丁处理乳脂率同对照组差异不显著(P>0.05),各处理组间牛乳乳蛋白、乳糖、非脂固形物差异不显著(P>0.05)。试验表明:芦丁能够提高奶牛泌乳性能,同时保持主要乳成分不受影响,在本试验条件下,芦丁适宜添加量为3.0mg/kg·BW。
试验二、芦丁在奶牛胃肠道不同部位出现的峰值研究
研究芦丁在奶牛胃肠道不同部位出现的峰值及其特点。利用4头安装有永久性瘤胃瘘和十二指肠瘘的成年奶牛,采用自身配对设计,对照期饲喂基础全混合日粮(TMR),试验期每日每头在基础日粮中一次性投喂芦丁3.0mg/kg·BW,在投喂后1 ~ 8 h每小时采集瘤胃液、十二指肠液和血液,检测其中芦丁和槲皮素的含量。结果显示,芦丁和槲皮素在奶牛瘤胃液中分别于饲喂后1 h和2 h达到峰值3.017±0.153μg/ml和1.148±0.153μg/ml;十二指肠液中在6 h出现峰值2.133±0.057μg/ml和0.214±0.004μg/ml;在血液中芦丁和槲皮素均出现两个峰值,芦丁的峰值出现在1 h(0.180±0.029μg/ml)和8 h(0.229±0.014μg/ml),槲皮素的峰值在1h(0.064±0.002μg/ml)和8h(0.089±0.007μg/ml)。试验表明:芦丁及其代谢产物槲皮素在奶牛胃肠道中出现的峰值因饲喂时间和消化部位不同而有差异。
试验三、芦丁对奶牛血液生化指标和激素水平的影响
在奶牛日粮中添加芦丁,测定奶牛血液生化指标、免疫指标和激素水平的变化,旨在探索芦丁对奶牛血清学指标和泌乳相关激素水平的影响。选用生理状态等相近的经产中国荷斯坦泌乳牛10头,随机分成2组,每组5头。分别在基础日粮中每日每头添加芦丁0.0 mg/kg·BW(对照组)和3.0 mg/kg·BW(试验组)。试验期11周,其中预饲期1周,正试期10周,在试验第5、35、65日采集血样,检测其生化、免疫和激素指标。试验组与对照组相比,总蛋白、白蛋白、葡萄糖水平差异不显著(P>0.05),芦丁可以显著降低奶牛血液中尿素氮的含量(P<0.05);IgG、谷丙转氨酶(ALT)、谷草转氨酶(AST)、碱性磷酸酶(ALP)、皮质醇(Cort)含量两组差异不显著(P>0.05),试验组溶菌酶(LZM)水平显著高于对照组(P<0.05);试验组和对照组奶牛血液中雌激素(E2)、孕激素(P)和催乳素(PRL)差异不显著(P>0.05),试验组催乳素水平由试验第5日12.19 ng/ml至第65日提高到13.94 ng/ml呈现上升趋势。试验显示:芦丁能够降低奶牛血液中尿素氮水平,提高溶菌酶含量。
试验四、芦丁对奶牛瘤胃消化代谢的影响
研究芦丁对奶牛瘤胃消化代谢的影响,旨在为探讨芦丁对奶牛瘤胃发酵的调控作用提供数据与参考。利用安装有永久性瘤胃瘘和十二指肠瘘的4头成年奶牛,采用自身配对设计,对照期饲喂TMR ,试验期每日每头在TMR中一次性投喂芦丁3.0mg.kg·BW,在投喂后1 h ~ 8 h每小时采集瘤胃液,检测瘤胃液中pH值、氨态氮(NH3-N)、挥发性脂肪酸(VFA)、瘤胃原虫数量、微生物蛋白的变化情况。结果显示,试验期瘤胃液pH值5.92低于对照期6.16(P<0.05),NH3-N浓度11.03 mg/100ml低于对照期20.07 mg/100ml(P<0.05),总挥发性脂肪酸(TVFA)试验期96.72 mmol/L高于对照期83.12 mmol/L(P<0.05),乙酸浓度61.00 mmol/L高于对照期51.47 mmol/L(P<0.05),丙酸浓度36.35 mmol/L高于对照期19.95 mmol/L(P<0.05),乙酸/丙酸的比例试验期与对照期差异不显著(P>0.05),乙酸、丙酸在TVFA中所占的比例试验期与对照期差异也不显著,试验期中瘤胃原虫数12.54(×105个/ml)低于对照期16.37(×105个/ml)(P<0.05),原虫蛋白含量试验期3.71 mg/ml低于对照期5.14 mg/m(lP<0.05),菌体蛋白含量试验期1.50 mg/ml高于对照期0.86 mg/ml(P<0.05)。试验表明:芦丁具有以下作用,降低瘤胃中NH3-N浓度,增加挥发性脂肪酸的总量,增加乙酸、丙酸的浓度,降低瘤胃pH值,抑制瘤胃原虫数量,降低原虫蛋白含量,增加瘤胃液中菌体蛋白的浓度。
试验五、体外法研究芦丁对奶牛瘤胃主要消化酶活性的影响
研究芦丁对奶牛瘤胃主要消化酶活性的影响。选择体况良好,体重600 kg左右,安装有永久性瘤胃瘘管的健康中国荷斯坦奶牛5头,采集瘤胃内容物制取固相和液相酶液,分别添加芦丁0.00 mg/ml(对照组)、0.02 mg/ml、0.04 mg/ml、0.06 mg/ml,混匀后检测其中淀粉酶、滤纸酶、β-葡萄糖苷酶、羧甲基纤维素酶、微晶纤维素酶、果胶酶、木聚糖酶的活性。结果显示,添加芦丁的试验组与对照组相比较,奶牛瘤胃内容物固相和液相总脱氢酶、滤纸酶、β-葡萄糖苷酶、羧甲基纤维素酶和微晶纤维素酶的活性均有显著差异(P<0.05),蛋白酶活性显著低于对照组(P<0.05),而淀粉酶、果胶酶与木聚糖酶与对照组相比较其酶活性差异不显著(P>0.05)。试验表明:芦丁能够提高奶牛瘤胃内容物固相和液相中总脱氢酶、滤纸酶、β-葡萄糖苷酶、羧甲基纤维素酶、微晶纤维素酶的活性。
试验六、体外法研究芦丁对牛初乳酶活的影响
利用奶牛分泌到初乳中的生物活性物质——酶作为指标,来分析和评价芦丁对奶牛乳腺细胞的作用。采集5头健康中国荷斯坦奶牛分娩后第二天的初乳,向初乳样品中添加5个芦丁浓度剂量梯度0 mol/L(对照)、10~(-7) mol/L、10~(-6) mol/L、10~(-5) mol/L、10~(-4) mol/L,混匀后检测初乳中β-N-乙酰氨基葡萄糖苷酶(NAG)、碱性磷酸酶(ALP)、乳过氧化物酶(LP)、γ-谷氨酰转肽酶(γ-GT)的活性。检测结果显示10~(-6) mol/L、10~(-5) mol/L、10~(-4) mol/L剂量组NAG酶比活力分别为7.61、6.92、6.43(U/L)显著低于对照组9.74(U/L)(P<0.05),10~(-7) mol/L剂量组NAG酶比活力为8.17 U/L与对照组相比差异不显著(P>0.05);各剂量组ALP酶活性差异不显著;10~(-7) mol/L、10~(-6) mol/L、10~(-5) mol/L、10~(-4) mol/L各剂量组LP酶比活力分别为58.30、65.00、72.94、77.03(U/ml)显著高于对照组48.89(U/ml)(P<0.05);10~(-7) mol/L、10~(-6) mol/L剂量组γ-GT酶比活力分别为11613.49、11371.1(6U/L),显著高于对照组10248.84(U/L)(P<0.05),10~(-5) mol/L、10~(-4) mol/L剂量组γ-GT酶比活力与对照组相比差异不显著(P>0.05)。本试验结果表明:在试验室条件下,芦丁能够提高奶牛初乳中LP和γ-GT酶活性,抑制NAG酶活性,对ALP影响差异不显著。
试验七芦丁对大鼠泌乳性能的影响
通过研究芦丁对大鼠泌乳性能、内分泌激素、乳腺器官指数的影响,考察芦丁调节大鼠乳腺泌乳和维持泌乳的作用。试验选择18只Wistar受孕母鼠,随机分为3组,每组6只,分别为对照组(每日每只灌胃生理盐水2ml)、芦丁组(每日每只灌服芦丁60 mg/kg·BW)、雌二醇组(每周每只肌注雌二醇60ug/kg·BW),从哺乳第4天开始连续给药2周,基础日粮相同。测定指标包括:大鼠泌乳量,仔鼠平均体增重,乳腺器官指数,血浆与乳腺组织中雌激素(E2)、孕激素(P)、催乳素(PRL)、生长激素(GH)的含量。试验结果显示,芦丁组大鼠泌乳量7.35(g/h)显著高于对照组5.73(g/h)(P<0.05),与雌二醇组8.22(g/h)差异不显著(P>0.05);芦丁组与雌二醇组相比,仔鼠体增重差异不显著(P>0.05),但芦丁组与雌二醇组仔鼠体增重显著大于对照组(P<0.05);芦丁组大鼠血浆与乳腺组织中E2、PRL、GH的水平显著高于对照组(P<0.05),低于雌二醇组(P<0.05),P水平各组间差异不显著(P>0.05);雌二醇组和芦丁组大鼠乳腺器官指数显著高于对照组(P<0.05)。试验结果表明:在本试验条件下,芦丁能够促进乳腺分泌乳汁,显著提高雌性大鼠泌乳量,对促进大鼠乳腺器官的发育有一定作用,能发挥类雌二醇样作用。
试验八芦丁对雌性青春期大鼠乳腺发育及相关激素与受体水平的影响
通过研究芦丁对雌性青春期大鼠内分泌激素水平、激素受体表达的影响和乳腺组织形态学变化的观察,探讨芦丁对乳腺发育的作用。选取24只雌性青春期Wistar大鼠,随机分为3组,对照组(Con):每日每只灌胃生理盐水2 ml;芦丁组(Rut):每日每只灌服芦丁60 mg/kg·BW;雌二醇组(Est):每周每只肌注雌二醇60μg/kg·BW。试验期2周,结束时检测大鼠血浆和乳腺组织中雌激素(E2)、孕激素(P)、催乳素(PRL)、生长激素(GH)含量及其受体表达,并进行乳腺组织形态学观察。研究结果显示,Est组、Rut组、Con组大鼠血浆与乳腺组织中E2、PRL水平分别依次降低,差异显著(P<0.05);Rut组P水平显著高于Con组(P<0.05),与Est组相比,差异不显著(P>0.05);Est组GH水平高于Rut组,Rut组GH水平高于Con组,差异均不显著(P>0.05)。雌激素受体(ER)、孕激素受体(PR)、催乳素受体(PRLR)、生长激素受体(GHR)在乳腺组织中的表达量依次为Est组>Rut组>Con组(P<0.05)。Rut组大鼠乳腺组织切片腺泡管腔直径大于Con组(P<0.05),小于Est组(P>0.05)。试验表明:在本试验条件下,芦丁能够提高雌性青春期大鼠E2、P水平,促进PRL分泌,上调乳腺组织中ER、PR、PRLR、GHR的表达量,促进乳腺发育,发挥类雌二醇样作用。
试验九芦丁对去卵巢处女大鼠乳腺发育作用的影响
研究芦丁对去卵巢处女大鼠内分泌激素水平、激素受体表达的影响以及乳腺组织形态变化,旨在了解和认识芦丁促进乳腺发育的类雌激素样作用功效。试验选择32只去卵巢处女Wistar大鼠,随机分为四个处理组,假手术组(Sham),每日每只灌胃2 mL生理盐水;卵巢切除组(Ova),每日每只灌胃2 mL生理盐水;去卵巢芦丁组(Ova+Rut),每日每只按60 mg/kg·BW剂量灌服芦丁;去卵巢雌二醇组(Ova+Est),每周每只肌注雌二醇(60 ug/kg·BW)一次。试验为期两周,试验结束后采集大鼠血液样品、乳腺组织样品进行测定分析。试验结果显示,Ova+Rut组和Ova+Est组大鼠血浆中雌激素(E2)的水平高于Ova组(P<0.05),该两组间差异不显著(P>0.05),Ova+Rut组水平低于Sham组(P<0.05),Ova+Est组与Sham组差异不显著(P>0.05),各处理组间乳腺组织中E2含量呈现相同规律;各处理组血浆与乳腺组织中催乳素(PRL)的含量变化趋势为Ova组
试验十芦丁对哺乳大鼠乳腺激素受体表达量的影响
本试验目的是研究芦丁对哺乳期大鼠泌乳相关激素受体表达量的影响。试验选择18只Wistar哺乳母鼠,随机分为3组,每组6只,分别为对照组(Lac-Con组)每日每只灌胃生理盐水(2 mL)一次、芦丁组(Lac-Rut组)每日每只灌服芦丁(60 mg/kg·BW)一次、雌二醇组(Lac-Est组)每周每只肌注雌二醇(60μg/kg·BW)一次,从哺乳第4天开始连续给药2周,基础日粮相同。检测哺乳大鼠乳腺组织中雌激素受体(ER)、孕激素受体(PR)、催乳素受体(PRLR)、生长激素受体(GHR)的表达量,并对乳腺组织进行形态学观察。试验显示,ER、PR、PRLR、GHR在乳腺组织中的表达量Lac-Est组>Lac-Rut组>Lac-Con组(P<0.05);乳腺腺泡/管腔直径统计显示Lac-Est组和Lac-Rut组高于Lac-Con组(P<0.05),Lac-Est组与Lac-Rut组差异不显著(P>0.05)。试验结果表明:在本试验条件下,芦丁具有诱导ER、PR、PRLR和GHR表达的作用。
综上所述,本文系统地研究了芦丁对奶牛生产性能的影响,对瘤胃消化代谢的调节作用,及对乳腺和机体免疫功能的影响,探讨了芦丁对乳腺发育和维持泌乳的影响及相关内分泌作用。根据试验结果与分析讨论,可以得出以下主要结论,在本研究条件下:芦丁及其代谢物槲皮素在瘤胃液中出现峰值的时间为饲喂后1 h和2 h,在十二指肠液中出峰时间为6 h,在血液中出双峰时间均为1 h和8 h。日粮中按3.0 mg/kg添加芦丁,可以显著提高奶牛产奶量;日粮中添加芦丁可以改善瘤胃发酵和氮代谢,促进氮的代谢和利用,在一定程度上可抑制瘤胃原虫数量,降低原虫蛋白含量,增加瘤胃液中菌体蛋白的浓度。日粮中添加芦丁能够提高奶牛瘤胃内容物固相和液相中总脱氢酶、滤纸酶、β-葡萄糖苷酶、羧甲基纤维素酶、微晶纤维素酶活性,对奶牛免疫功能和激素水平无调节作用,可在一定程度上提高血清中溶菌酶的浓度,降低血液中尿素氮水平。芦丁能够提高奶牛初乳中LP和γ-GT酶活性,抑制NAG酶活性,对ALP影响差异不显著。在Wistar大鼠试验中,芦丁能够促进哺乳大鼠乳腺分泌乳汁,对大鼠乳腺器官的发育有一定的促进作用,能发挥类雌二醇样作用;能够提高雌性青春期大鼠E2、P水平,促进PRL分泌,上调乳腺组织中ER、PR、PRLR、GHR的表达量,促进乳腺发育,有类雌二醇样作用的功效;能够提高去卵巢处女大鼠E2水平,促进垂体分泌PRL、GH,上调乳腺组织中ER、PRLR、GHR的表达量;具有诱导ER、PR、PRLR和GHR表达的作用。
Rutin is the flavonoids extracted from the plant, and is widely distributed in nature. Studies have shown that rutin can be used not only in treatments but also as health care products for humans. From perspectives of digestive physiology and the physiology of lactation, the performance of dairy cows depends to a large extent on the rumen digestion and metabolism of nutrients and the development of lactating mammary gland.
In this series of researches, dairy cows and Wistar rats were used as animal models to study the effects of rutin supplementation on the production performance of diary cows, internal environment, digestion and metabolism of rumen, and the development and lactation maintenance of mammary gland. Besides, the action mechanisms and using effects of rutin on dairy cows physiological nutrition and mammary gland development were explored from aspects of nutriology and physiology. The present research consists of ten experiments which were listed as follows:
EXP 1. Effect of Rutin Supplementation on Lactation Performance
This study was designed to investigate the effects of rutin supplementation on milk yield in dairy cows, to find the optimum dosage of rutin and subsequently to offer the scientific basis for the usage of rutin in cows nutrition and feed. Twenty cows in their peak lactation were randomly divided into 4 groups and each was offered a basal diet supplemented with 0, 1.5, 3.0 or 4.5 mg rutin/kg·BW of diet. The trial lasted 11 weeks, including an adaption period of one week and a ten-week experimental period. Milk yields were recorded every other day, and milk compositions were analyzed on the 5th, 35th and 65th day. The experiment showed that, compared to the control group, the cows receiving 3.0 and 4.5 mg·rutin/kg·BW had significantly higher milk yield (P<0.05). No significant difference in milk yield was detected between the cows receiving 1.5 mg·rutin/kg·BW and the control group (P > 0.05). The milk fat content of the cows receiving 1.5 or 3.0 mg·rutin/kg·BW was lower than that of the control group (P<0.05). No significant difference in the milk fat content was observed between the cows offered 4.5 mg·rutin/kg·BW and the control group (P>0.05). The contents of milk protein, lactose and milk non-fat solid matter did not differ significantly among groups (P>0.05). Experiments indicated that dietary rutin improved the production performance of dairy cows,while the main milk compositions were not affected. Under this experimental conditions, the appropriate dosage for rutin supplementation was 3.0 mg/kg·BW diet.
EXP 2. Study on the Peak of Rutin in Different Parts of the Gastrointestinal Tract in Dairy Cows
4 adult cows with permanent rumen fistula and duodenal cannulae were attributed to a self control design and used to investigate the time and characteristics of peak occurrence in different parts of the gastrointestinal tract in dairy cows. Total mixed rations ( TMR ) were used as a basal diet in control period. Cows were fed with rutin one at a time per day in test period. The levels of rutin and quercetin in rumen fluid, duodenal content and blood were detected once per hour from 1 h to 8 h after feeding. The results showed that the concentration of rutin and quercetin in rumen fluid reached their peak levels at 1 h ( 3.017±0.153μg/ml ) and 2 h ( 1.148±0.153μg/ml ) respectively. The peak level in duodenal content occurred at 6h ( 2.133±0.057μg/ml and 0.214±0.004μg/ml ). Rutin had two peaks in blood at 1 h ( 0.180±0.029μg/ml )and 8 h(0.229±0.014μg/ml)respectively. So did quercetin at 1h(0.064±0.002μg/ml )and 8h(0.089±0.007μg/ml). These results indicated that feeding time and digest positions affected the peak occurrence of rutin and aglycone quercetin in the gastrointestinal tract in dairy cows.
EXP 3. Effects of Rutin Supplementation on Blood Metabolites and Levels of Hormone in Dairy Cows
By investigating the changes of blood metabolites and levels of hormone in dairy cows after rutin supplementation, we try to study the effects of rutin on immune function and milk-related hormones. Ten cows in their peak lactation were randomly divided into 2 groups and each was offered a basal diet supplemented with 0.0 and 3.0 mg rutin/kg·BW of diet. The trial was 11 weeks, including an adaption period of one week and a ten-week experimental period. The vein blood samples were analyzed on the 5th, 35th and 65th day. Test serum biochemical indexes, immune indexes and lactation-related hormones. Compared to the control group, no significant difference was observed in the serum concentration of TP, ALB and Cort (P>0.05). But rutin could reduce the content of urea nitrogen in blood (P<0.05).There was no significant difference in the serum concentration of IgG, ALT, AST, ALP and cortisol between the cows receiving 3.0 mg·rutin/kg·BW and the control group (P<0.05). The lysozyme content of the experimental group was higher than that of control group (P<0.05). The serum concentration of estrogen (E2) and progestin (P) did not differ significantly between the treatments (P>0.05), but the serum progesterone level which increased from 12.19 ng/ml on the 5th day to 13.94 ng/ml on the 65th day showed an upward trend in rutin-supplemented cows compared to control. It suggested that rutin could decrease blood urea nitrogen and increase serum lysozyme content.
EXP 4. Effects of Rutin on the Ruminal Digestibility and Metabolism of Dairy Cows
The study was designed to investigate the role of rutin on the ruminal digestibility and metabolism of dairy cows, and subsequently to provide data and references to exploring the effect of rutin on ruminal fermentation. 4 adult cows with permanent rumen fistula and duodenal cannulae were attributed to a self control design and used in the investigation. TMR were used as basal diet in the control period. Cows were fed with rutin one at a time per day in the test period. The levels of pH, ammonia nitrogen ( NH3-N ), volatility fatty acid ( VFA ), the number of rumen protozoa and microbial protein ( MCP ) were detected once per hour from 1 h to 8 h after feeding. The results showed that the rumen fluid pH in the test period ( 5.92 ) was lower than that in the control period ( 6.16 ) (P<0.05). The concentration of NH3-N in the test period ( 11.03 mg/100ml ) was less than that in the control period ( 20.07 mg/100ml ) (P<0.05).Compared to the control period( 83.12 mmol/L ) , the concentration of TVFA in the test period ( 96.72 mmol/L ) was higher (P<0.05). Acetic acid in the test period ( 61.00 mmol/L ) was on a higher level compared to the control period ( 51.47 mmol/L ) (P<0.05) and so was propionic acid in the test period ( 36.35 mmol/L ) compared to the control period ( 19.95 mmol/L ) (P<0.05). There were no significant difference in both the proportion of acetic acid and propionic acid in TVFA (P<0.05). The number and protein content of rumen protozoa in the test period ( 12.54×10~5个/ml, 3.71 mg/ml ) were less than those in the control period ( 16.37×10~5个/ml , 5.14 mg/ml ) (P<0.05). In the test period the MCP ( 1.50 mg/ml ) was higher compared to the control period (0.86 mg/ml ) (P<0.05). Those results indicated that rutin could reduce the concentration of NH3-N , increase the TVFA and the concentration of acetic acid and propionic acid, lower pH level, bring down the number and the protein content of rumen protozoa, and increase MCP in the rumen.
EXP 5. Study the Effect of Rutin on Activities of Digestive Enzymes in the Rumen in Vitro
This study was conducted to investigate the effect of rutin on activities of digestive enzymes in the rumen of dairy cows. 5 healthy about 600 kg-weight China Holstein Cows with permanent rumen fistula were used in this study. Rutin was added into the enzyme ( solid and liquid fraction) extracted from rumen content on the dosage of 0.00 mg/ml (control group), 0.02 mg/ml, 0.04 mg/ml, 0.06 mg/ml respectively and. After mixing, the enzyme activities of protease, amylase, total dehydrogenase, filter paper enzyme,β-glucosidase, carboxymethyl cellulose (CMC), avicelase, pectinase, xylanase were measured. The results showed that compared to the control group, the enzyme activities total dehydrogenase, filter paper enzyme,β-glucosidase, CMC, avicelase in experimental groups significantly differed in both liquid and solid fraction ( P<0.05 ). The enzyme activities of protease were lower than control group ( P<0.05 ). There were no significant difference in the activities about amylase, pectinase and xylanase( P>0.05). In all groups, the enzyme activities of solid fraction were significantly higher than that of liquid faction. It suggested that rutin could enhance the enzyme activities of filter paper enzyme,β-glucosidase, CMC, avicelase in solid and liquid fraction in the rumen of dairy cows. The value of the enzyme activities in solid fraction was higher than that in liquid fraction.
EXP 6. Study the Effect of Rutin on the Enzyme Activities of Bovine Colostrum in Vitro
The enzyme ( the bioactive substances ) secreted into colostrum by dairy cows was used as an indicator to analyze and evaluate the action of rutin on cow mammary cells. The colostrum samples of five healthy Chinese Holstein cows was collected the day after delivery. Rutin was added into the samples on the dosage of 0(control)、10~(-7)、10~(-6)、10~(-5)、10-(4mol/L ). After mixing, the enzyme activities ofβ-N-acetylglucosaminidase ( NAG ), alkaline phosphatase ( ALP ), lactoperoxidase ( LP ),γ-glutamyl transpeptidase (γ-GT ) were measured. Results showed that the enzyme activites of NAG in 10~(-6)、10~(-5)、10~(-4)(mol/L )groups ( 7.61、6.92、6.43 U/L respectively)were lower than that in the control group 9.74(U/L)(P<0.05). No significant difference was observed between 10~(-7)(mol/L)group (8.17 U/L) and the control group (P>0.05). The enzyme activities of ALP did not differ significantly among the treatments (P>0.05). The enzyme activities of LP in 10~(-7)、10~(-6)、10~(-5)、10~(-4)(mol/L)groups ( 58.30、65.00、72.94、77.03 U/ml respectively) were higher than the control group ( 48.89 U/ml )(P<0.05). The enzyme activities ofγ-GT in 10~(-7)、10~(-6)(mol/L)groups (11613.49、11371.16U/L respectively)were higher than 0(mol/L)(10248.84U/L)(P<0.05), but there were no significant differences in 10~(-5)、10~(-4)(mol/L)groups 10036.74、9542.33U/L) ompared to the control group(P<0.05). This result suggested that, under such experimental conditions, rutin could improve the enzyme activities of LP andγ-GT, inhibit the enzyme activities of NAG, and exert no significant effect on the enzyme activities of ALP.
EXP 7. Effect of dietary rutin on laction performance in rats
The objective of this study was to investigate the regulatory effect of rutin on lactation of rats by examining the effects of rutin on milk yields, endocrine hormones and mammary organ indexes of rats. Eighteen pregnant Wistar rats were randomly assigned to 3 treatments: control, gastric infusion of rutin at 60 mg/kg·BW per day, and intramuscular injection of estradiol 60μg/kg·BW weekly from the fourth lactation day for two weeks with the same basal diet. The milk yield of rats, body weight gain of the neonatal rats, mammary gland index, the level of estrogen (E2), progesterone (P), prolactin (PRL) and growth hormone (GH) in plasma and gland tissues were measured. The results shown that the milk yield of the rats receiving rutin ( 7.35 g/h ) was higher than that of the control group ( 5.73 g/h ) (P<0.05), but no obvious difference was observed when compared to the estradiol group ( 8.22 g/h ) (P>0.05). The body weight gain of the neonatal rats in the rutin group had no distinct difference with that in the estradiol group (P<0.05), but these two groups were higher than the control group (P>0.05). The level of E2, PRL and GH in plasma and gland tissues of the rats in the rutin group was higher than that in the control group (P<0.05), but less than that in the estradiol group (P<0.05), and the differences of P level among groups were not distinct (P>0.05). The indexes of mammary gland of the rats in the estradiol and rutin group were higher than that in the control group (P<0.05). Those results indicated that, under such experimental conditions, dietary rutin could promote mammary gland to secrete milk , significantly improve the milk yield of female rats, affect to some extent the development of the thymus, and excert E2-like effects.
EXP 8. Effects of Rutin on the Development of Mammary Gland and Levels of Relevant Hormones and Hormonal Receptors of Pubertal Female Rats
The objective of this study was to investigate the regulatory effect of rutin on mammary glands of unmarried rats by studying the plasma level of endocrine hormones, the expression of hormonal receptors and the changes in the histomorphology of mammary tissues. Twenty four intact virgin rats were randomly assigned to three treatments: 1) gastric infusion of 2 ml normal saline per mouse per day (control (Con)); 2) gastric infusion of 60 mg rutin/kg·BW per day (Rutin (Rut)); and 3) intramuscular injection of 60μg estradiol/kg·BW weekly (Estradiol (Est)). The trial lasted two weeks. The concentration of estrogen (E2), progesterone (P), prolactin (PRL) and growth hormone (GH) in plasma and gland tissues was determined, and the expression of estrogen receptors (ER), progesterone receptors (PR), prolactin receptors (PRLR) and growth hormone receptors (GHR) in gland tissues was measured. The histomorphology of mammary tissues was also monitored. Results showed that the concentration of E2 and PRL in plasma and mammary tissues was ranked as Est>Rut>Con, and the differences were distinct (P<0.05), the concentration of P in plasma and mammary tissues in Rut group was higher than that in Con group(P<0.05), and there was no difference between Rut and Est groups(P>0.05).The concentration of GH in Est grouo was higher than that in Rut group, so was Rut group compared to Con group, and the difference was not significant(P>0.05). The expression of ER, PR, PRLR and GHR in mammary tissues was ranked as Est>Rut>Con (P<0.05). The diameter of mammary acinar cells and lumen was higher (P<0.05) in Rut group than that in Con group, but lower than that in Est group (P>0.05). These results suggested that, under such experimental conditions, rutin could improve the plasma concentration of E2 and P in pubertal female rats, promote PRL release, and up-regulate the expression of ER, PR, PRLE and GHR, exert E_2-like effects.
EXP 9. Experiment Study of Rutin on Mammary Gland Development in Ovariectomized Virgin Rats
The objective of this study was to realize the estrogen-like effect of rutin on promoting mammary gland development by examining the levels of endocrine hormones , the expression of hormonal receptor and the changes in the histomorphology of mammary tissues of ovariectomized virgin rats. Thirty-two ovariectomized virgin rats were randomly assigned to four treatments: sham, gastric infusion of 2 ml normal saline per mouse and per day; Ova, gastric infusion of 2 ml normal saline per mouse and per day; Ova+Rut, gastric infusion of 60 mg rutin/kg·BW per day; Ova+Est, intramuscular injection of 60μg estradiol/kg·BW weekly. The trial lasted two weeks. The samples of blood, gland tissues, thymuses and spleens were collected for quantitative analysis. The level of estrogen (E2), prolactin (PRL) and growth hormone (GH) in plasma and gland tissues were detected and the expression of estrogen receptor (ER), prolactin receptor (PRLR) and growth hormone receptor (GHR) in gland tissues were detected. Histomorphology of development about mammary gland were observed. The results showed that, the level of E2 in plasma in Ova+Rut and Ova+Est were higher than that in Ova (P<0.05),and the difference between these two groups was not significant (P> 0.05), Ova+Rut was lower than Sham (P<0.05), Ova+Est and Sham had no distinct difference(P>0.05). The level of E2 in gland tissues showed the same trend among different treatment groups. The order of the PRL in plasma and gland tissues was Ova
EXP 10. Effect of Dietary Rutin on the Expression of Hormonal Receptor in Lactating Rats
The objective of this study was to observe effects of rutin on mammary gland development, endocrine hormone level and hormonal receptor expression in lactation rats. Eighteen Wistar rats were randomly assigned to three treatments: control (Lac-Con), gastric infusion of 2 ml normal saline per mouse and per day. Rutin (Lac-Rut), gastric infusion of 60 mg rutin/kg·BW per day. Estradiol (Lac-Est), intramuscular injection of 60μg estradiol/kg·BW weekly. The experiment lasted for two weeks from the fourth lactation day with the same basal diet. The expression of estrogen receptor (ER), progesterone receptor (PR), prolactin receptor (PRLR) and growth hormone receptor (GHR) in gland tissues were measured. Histomorphology of mammary gland development were observed. The results showed that the expression of ER, PR, PRLR and GHR in gland tissues was Lac-Est > Lac-Rut > Lac-Con (P<0.05). The diameter of mammary gland alveolus showed that Lac-Est and Lac-Rut were higher than Lac-Con (P<0.05), there was no significant difference between Lac-Est and Lac-Rut (P>0.05). The results indicated that, under such experimental conditions, rutin had the function of inducing the expression of ER, PR, PRLE and GHR.
To sum up, in this dissertation, we systematically studied the effect of rutin on production performance of dairy cows, the regulating action of rutin on rumen digestion and metabolism, and the influence of rutin on the immune function of the mammary gland and the organism. We also explored the role of rutin on mammary gland development and the maintenance of lactating as well as the mechanisms of related endocrine system. The following main conclusions could be drawn according to the experimental results, analysis and discussions. Under the study conditions rutin had the functions as follows:
The peak time of rutin and its metabolite– quercetin in the rumen fluid occurred at 1h and 2 h after feeding respectively, in the duodenal content the peak time both occurred at 6 h, and in the blood there were two peaks which both occurred at 1h and 8 h. Dietary rutin added on the dosage of 3.0 mg/kg·BW could obviously promote the milk yield and no significant difference was observed in the milk composition. Dietary rutin could improve rumen fermentation and the nitrogen metabolism, promote the nitrogen metabolism and utilization, bring down the number and the protein content of rumen protozoa to some extent, and increase MCP in the rumen. Dietary rutin could enhance the enzyme activities of total dehydrogenase, filter paper enzyme,β-glucosidase, CMC, avicelase in solid and liquid fraction in the rumen of dairy cows, but had no regulatory role on the immune function and hormone level. It could also increase serum lysozyme content to some degree, and decrease blood urea nitrogen level. Rutin could improve the enzyme activities of LP andγ-GT, inhibit the enzyme activities of NAG, and exert no significant effect on the enzyme activities of ALP in the colostrum of dairy cows. In the experiments using rats as animal model, rutin could promote mammary glands of pregnant rats to secrete milk, have some effect on the development of the rat’s thymus, and excert E2-like effects. Rutin could also increase the level of E2 and P in pubertal female rats, promote PRL release, and up-regulate the expression of ER, PR, PRLE and GHR, promote the development of mammary glands, and exert E2-like effects; Rutin could increase the level of E2 in ovariectomized virgin rats, promote pituitary PRL and GH release, up-regulated the expression level of ER, PRLE and GHR; It could also increase the level of E2 in ovariectomized virgin rats, promote pituitary PRL and GH release, up-regulated the expression level of ER, PRLE and GHR, and have the function of inducing the expression of ER, PR, PRLE and GHR.
引文
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