不同日粮模式对泌乳奶牛乳腺乳蛋白合成影响的研究
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摘要
牛乳中乳蛋白含量较低是中国乳品行业所面临的重要问题，立足于中国的饲料资源，通过日粮调控的途径提高乳蛋白含量与产量将有现实意义。本研究比较了中国奶牛养殖模式中三种具有代表性的日粮模式对泌乳奶牛乳腺乳蛋白合成的影响，找出了可能影响乳蛋白合成的关键因素，为提高奶业生产水平，推动奶业发展提供科学数据。
试验1不同氨基酸模式对奶牛乳腺上皮细胞酪蛋白合成的影响及机理研究
本论文首先在体外研究了不同氨基酸模式对奶牛乳腺上皮细胞酪蛋白合成的影响，目的是确定氨基酸模式是否会影响乳腺上皮细胞酪蛋白的分泌，并对其机理进行初步研究。试验采用完全随机试验设计，氨基酸模式分别为：低蛋白日粮条件下血液氨基酸组成模式组(LPBP)、全乳蛋白氨基酸组成模式组(MP)、酪蛋白氨基酸组成模式组(CP)、80%酪蛋白+20%乳清蛋白氨基酸组成模式组(CLP)，每个处理3个重复，试验重复3遍（n=12）。用RT-qPCR检测CSN1S1、CSN3、GLUT1、PRLR、STAT5和mTOR基因表达量，用Elisa法检测αS-酪蛋白合成量，用CCK-8检测细胞增殖活性。试验结果显示：氨基酸模式的不同影响奶牛乳腺上皮细胞酪蛋白的合成，全乳蛋白模式可能是种较为理想的氨基酸模式。氨基酸模式通过调节mTOR、STAT5、PRLR和GLUT1基因的表达影响酪蛋白的合成。此外，研究发现在体外培养条件下，乳腺上皮细胞增殖活性与乳蛋白基因表达的趋势并不致。
试验2不同日粮模式对泌乳奶牛乳产量与乳成分的影响
论文的第二部分通过动物试验明确了不同日粮模式对泌乳奶牛乳产量与乳成分的影响程度，为进步阐述不同日粮模式对乳腺内乳蛋白合成的影响提供数据。采用完全随机区组试验设计，30头牛分为3组，每组10头。分别饲喂三种日粮：1）代表规模化养殖场日粮模式的混合粗饲料组（Mixed forage MF）；2）代表奶牛养殖小区养殖模式的高精料秸秆组（Corn straw A）；3）代表散户养殖模式的低精料秸秆组（Corn straw B）。试验期12周，预饲期3周,正式试验期9周，每3周为个试验周期，在每个试验周期的15～18d采集饲料样、乳样，并记录产奶量。试验结果显示：MF组日粮的整体生产水平最佳，CSA组虽然在乳蛋白含量与产量上与MF组没有显著性差异，但是泌乳效率显著低于MF组，CSB组除乳脂率外，其他乳成分指标均低于CSA组与MF组。以上结果提示：营养水平满足需要时，平衡日粮的营养素利用效率更高；CSB组日粮营养水平满足不了泌乳需要。
试验3不同日粮模式下泌乳奶牛动静脉血中乳蛋白合成相关代谢物分析
在试验2的基础上，对泌乳奶牛阴部外动脉与乳静脉血中乳蛋白合成相关代谢物含量进行分析，以期找出影响乳蛋白合成的关键因素。试验结果显示：不同日粮模式对泌乳奶牛阴外动脉血液中PRL、GH、INR、IGF-1、和葡萄糖的含量没有显著影响（P>0.05），CSA组阴外动脉血中血浆尿素氮(BUN)及瘦素含量显著高于CSB组与MF组（P <0.05），提示高精料秸秆组氮的利用效率较低，瘦素可能影响了CSA组乳脂的合成。CSA组和CSB组乳静脉静脉血中谷草转氨酶（AST）含量显著高于MF组(P <0.05),提示秸秆组日粮可能会影响肝脏的健康。不同日粮模式下泌乳奶牛阴外动脉血浆中大部分氨基酸浓度与比例没有显著差异，但是Asp、Arg、Thr、His、Gly和Leu的比例有显著差异或有差异显著的趋势。
试验4不同日粮模式下泌乳奶牛乳腺对氨基酸的摄取与利用
在试验2的基础上，分析了不同日粮模式下乳腺对氨基酸的摄取与利用的差异。结果显示：限制CSB组乳腺对氨基酸利用的因素是阴外动脉血所提供EAA的绝对量少，其原因是CSB组日粮营养水平低，血流量小，所提供营养物质的绝对量不能完全满足乳腺泌乳的需要。CSA组与MF组乳腺对氨基酸的吸收与利用方面没有显著的差别。
试验5不同日粮模式下泌乳奶牛乳腺组织中泌乳相关基因表达
在试验2的基础上，采集了不同日粮模式下泌乳奶牛的乳腺组织用于RNA提取，并用RT-qPCR检测CSN1S1、CSN1S2、CSN3、INSR、PRLR、GHR、JAK2、STAT5和mTOR基因表达量。研究结果显示：日粮模式的不同能够影响泌乳奶牛乳腺组织内乳蛋白基因的表达，日粮营养水平与平衡性会通过影响乳腺组织内PRLR、GHR、INSR、及mTOR基因的表达，进而影响乳蛋白的合成。在营养水平相同的情况下，平衡的日粮更能促进乳蛋白基因的表达。
总体试验结果显示：氨基酸供给量与在乳腺内利用效率的差异是影响不同日粮模式下乳蛋白含量与产量的重要原因，通过检测乳腺组织中泌乳相关基因的表达量可以反映出不同日粮模式下泌乳奶牛乳腺内乳蛋白的合成效率；氨基酸模式的不同能够影响奶牛乳腺上皮细胞酪蛋白的合成，全乳蛋白模式可能是种较为理想的氨基酸模式。
Low milk protein content is the issue for producers in China, so based on the feedresources of China, formulate diets is of practical significance as a means to improvemilk protein production.This study was to evaluate the effects of three diets on the milkprotein synthesis in mammary gand of dairy cows, and those diets is very representativeof feeding for dairy cows in China. Identify key factors that may affect the milk proteinsynthesis, and to provide scientific data in order to increase the level of milk production,promote the development of dairy industry.
Experiment1: Study on the effects of Different Amino Acid Pattern on caseinsynthesis in Primary Cultured Bovine Mammary Epithelial Cells
A completely random design was used. Four AA patterns reatments are as follows:blood AA pattern in low protein diet (LPBP); milk protein AA pattern (MP);80%caseinplus20%lactoalbumin AA pattern (CLP); casein AA pattern (CP). Each treatment hasthree replicates, and all experiments were repeated three times. The expression ofCSN1S1, CSN3, GLUT1, PRLR, STAT5, mTOR genes and the content of αS-casein weredetermined by RT-QPCR method and αS-casein ELISA Kit, respectively.Cellproliferation was determined by CCK-8method.The results showed that: The milkprotein AA pattern might increase major milk protein genes expression. Perhaps, anappropriate AA pattern can promote mTOR STAT5PRLR GLUT1genes expression, andresult in increased milk protein synthesis. In addition, the level of geng expression trendwas different from the proliferation in mammary gland epithelial cell.
Experiment2：The effects of Different diets on milk production and milkcomposition of dairy cows
The second part was mainly conducted to study the effects of three diets on milkproduction and milk composition of dairy cows, and provide the data to the research ofmilk protein synthesis in the mammany gland. Thirty multiparous Holstein cows wereassigned to a single factor block design and cows were divided into three groups.Threediets were:1）Mixed forage represent Large-scale dairy farming.2) High-concentrate andcorn stover represent dairy farming.3) Low-concentrate and corn stover represent retaildairy farming.The exprement period were12weeks, and pre-feeding period were3weeks, every3weeks for a test cycle.Feed and milk sample were collected at15～18d ineach cycle.The results showed that: The MF group had a best performance, there was no difference between CSA and MF in milk protein yield and milk proten content, however,the Lactation efficiency was higher in MF vs.CSA. Compared to the CSA and MF, CSBhad lower level of all indicators of milk composition except milk fat. The results suggestthat, blance diets more efficient in nutrients metabolism, the energy level of CSB can notmaintain the lactating.
Experiment3: The effects of Different diets on metabolin content related to milkprotein synthesis in artery/vein blood of dairy cows
Based on experiment2, the objective of this part was to evaluate the metabolincontent related to milk protein synthesis in artery/vein blood of dairy cows and found outthe key factor that affect milk protein synthesis.The results showed that:PRL,GH,INR,IGF-1and glouse content in artery/vein blood have no difference indifferent diets(P>0.05), the level of BUN and leptin in EPA were higher in CSAvs.CSB and MF significantly, compare to the MF, CSA and CSB were significant higherin AST. Most of the amino acids have no difference in different diets, but, the ratio ofAsp, Arg, Thr, His, Gly and Leu have significant difference or difference in the trend.The results suggest that, CSA and CSB have low efficiency of nitrogen utilization. Leptinmay affect the synthesis of milk fat in the CSA group. The health of the liver wasaffented by CSA and CSB, perhaps.
Experiment4: The effects of Different diets on AA uptake and utilization inmamany gland of dairy cows
Based on experiment2, the objective of this part was to evaluate the difference ofAA uptake and utilization in mammary gland.The results showed that: the AA utilizationof mammary gland of CSB was restricted by the level of EAA in EPA, the reason was thelow level of nutrition and blood flow. There was no difference in AA uptake andutilization between CSA and MF.
Experiment5: Effects of different diets on the Gene Expression Related to MilkProtein Synthesis in the Mammary Gland of dairy cows
Based on experiment2, Mammary gland tissue (ca.500mg/animal) for RNAextraction were randomly performed for half numbers of cows in each group aftermilking on the last day of experiment. The expression of CSN1S1, CSN1S2,CSN3,INSR,PRLR, GHR1A, JAK2，STAT5and mTOR genes were determined by RT-QPCR method.The results showed that: Different diets affected the efficiency of milk protein synthesisin lactating Holstein cows. The balance dites can promote PRLR GHR1A and mTORgenes expression, resulting in increased milk protein synthesis.
The final results showed that: AA supply and utilization efficiency in mammarygland result in different milk protein and yield, milk protein synthesis efficiency wasevaluated by the gene expression related to milk protein synthesis in the mammary glandtissue. The milk protein AA pattern might increase major milk protein genes expression.Perhaps, the milk protein pattern can promote milk protein synthesis in mammary glandepithelial cell better.
试验1不同氨基酸模式对奶牛乳腺上皮细胞酪蛋白合成的影响及机理研究
本论文首先在体外研究了不同氨基酸模式对奶牛乳腺上皮细胞酪蛋白合成的影响，目的是确定氨基酸模式是否会影响乳腺上皮细胞酪蛋白的分泌，并对其机理进行初步研究。试验采用完全随机试验设计，氨基酸模式分别为：低蛋白日粮条件下血液氨基酸组成模式组(LPBP)、全乳蛋白氨基酸组成模式组(MP)、酪蛋白氨基酸组成模式组(CP)、80%酪蛋白+20%乳清蛋白氨基酸组成模式组(CLP)，每个处理3个重复，试验重复3遍（n=12）。用RT-qPCR检测CSN1S1、CSN3、GLUT1、PRLR、STAT5和mTOR基因表达量，用Elisa法检测αS-酪蛋白合成量，用CCK-8检测细胞增殖活性。试验结果显示：氨基酸模式的不同影响奶牛乳腺上皮细胞酪蛋白的合成，全乳蛋白模式可能是种较为理想的氨基酸模式。氨基酸模式通过调节mTOR、STAT5、PRLR和GLUT1基因的表达影响酪蛋白的合成。此外，研究发现在体外培养条件下，乳腺上皮细胞增殖活性与乳蛋白基因表达的趋势并不致。
试验2不同日粮模式对泌乳奶牛乳产量与乳成分的影响
论文的第二部分通过动物试验明确了不同日粮模式对泌乳奶牛乳产量与乳成分的影响程度，为进步阐述不同日粮模式对乳腺内乳蛋白合成的影响提供数据。采用完全随机区组试验设计，30头牛分为3组，每组10头。分别饲喂三种日粮：1）代表规模化养殖场日粮模式的混合粗饲料组（Mixed forage MF）；2）代表奶牛养殖小区养殖模式的高精料秸秆组（Corn straw A）；3）代表散户养殖模式的低精料秸秆组（Corn straw B）。试验期12周，预饲期3周,正式试验期9周，每3周为个试验周期，在每个试验周期的15～18d采集饲料样、乳样，并记录产奶量。试验结果显示：MF组日粮的整体生产水平最佳，CSA组虽然在乳蛋白含量与产量上与MF组没有显著性差异，但是泌乳效率显著低于MF组，CSB组除乳脂率外，其他乳成分指标均低于CSA组与MF组。以上结果提示：营养水平满足需要时，平衡日粮的营养素利用效率更高；CSB组日粮营养水平满足不了泌乳需要。
试验3不同日粮模式下泌乳奶牛动静脉血中乳蛋白合成相关代谢物分析
在试验2的基础上，对泌乳奶牛阴部外动脉与乳静脉血中乳蛋白合成相关代谢物含量进行分析，以期找出影响乳蛋白合成的关键因素。试验结果显示：不同日粮模式对泌乳奶牛阴外动脉血液中PRL、GH、INR、IGF-1、和葡萄糖的含量没有显著影响（P>0.05），CSA组阴外动脉血中血浆尿素氮(BUN)及瘦素含量显著高于CSB组与MF组（P <0.05），提示高精料秸秆组氮的利用效率较低，瘦素可能影响了CSA组乳脂的合成。CSA组和CSB组乳静脉静脉血中谷草转氨酶（AST）含量显著高于MF组(P <0.05),提示秸秆组日粮可能会影响肝脏的健康。不同日粮模式下泌乳奶牛阴外动脉血浆中大部分氨基酸浓度与比例没有显著差异，但是Asp、Arg、Thr、His、Gly和Leu的比例有显著差异或有差异显著的趋势。
试验4不同日粮模式下泌乳奶牛乳腺对氨基酸的摄取与利用
在试验2的基础上，分析了不同日粮模式下乳腺对氨基酸的摄取与利用的差异。结果显示：限制CSB组乳腺对氨基酸利用的因素是阴外动脉血所提供EAA的绝对量少，其原因是CSB组日粮营养水平低，血流量小，所提供营养物质的绝对量不能完全满足乳腺泌乳的需要。CSA组与MF组乳腺对氨基酸的吸收与利用方面没有显著的差别。
试验5不同日粮模式下泌乳奶牛乳腺组织中泌乳相关基因表达
在试验2的基础上，采集了不同日粮模式下泌乳奶牛的乳腺组织用于RNA提取，并用RT-qPCR检测CSN1S1、CSN1S2、CSN3、INSR、PRLR、GHR、JAK2、STAT5和mTOR基因表达量。研究结果显示：日粮模式的不同能够影响泌乳奶牛乳腺组织内乳蛋白基因的表达，日粮营养水平与平衡性会通过影响乳腺组织内PRLR、GHR、INSR、及mTOR基因的表达，进而影响乳蛋白的合成。在营养水平相同的情况下，平衡的日粮更能促进乳蛋白基因的表达。
总体试验结果显示：氨基酸供给量与在乳腺内利用效率的差异是影响不同日粮模式下乳蛋白含量与产量的重要原因，通过检测乳腺组织中泌乳相关基因的表达量可以反映出不同日粮模式下泌乳奶牛乳腺内乳蛋白的合成效率；氨基酸模式的不同能够影响奶牛乳腺上皮细胞酪蛋白的合成，全乳蛋白模式可能是种较为理想的氨基酸模式。
Low milk protein content is the issue for producers in China, so based on the feedresources of China, formulate diets is of practical significance as a means to improvemilk protein production.This study was to evaluate the effects of three diets on the milkprotein synthesis in mammary gand of dairy cows, and those diets is very representativeof feeding for dairy cows in China. Identify key factors that may affect the milk proteinsynthesis, and to provide scientific data in order to increase the level of milk production,promote the development of dairy industry.
Experiment1: Study on the effects of Different Amino Acid Pattern on caseinsynthesis in Primary Cultured Bovine Mammary Epithelial Cells
A completely random design was used. Four AA patterns reatments are as follows:blood AA pattern in low protein diet (LPBP); milk protein AA pattern (MP);80%caseinplus20%lactoalbumin AA pattern (CLP); casein AA pattern (CP). Each treatment hasthree replicates, and all experiments were repeated three times. The expression ofCSN1S1, CSN3, GLUT1, PRLR, STAT5, mTOR genes and the content of αS-casein weredetermined by RT-QPCR method and αS-casein ELISA Kit, respectively.Cellproliferation was determined by CCK-8method.The results showed that: The milkprotein AA pattern might increase major milk protein genes expression. Perhaps, anappropriate AA pattern can promote mTOR STAT5PRLR GLUT1genes expression, andresult in increased milk protein synthesis. In addition, the level of geng expression trendwas different from the proliferation in mammary gland epithelial cell.
Experiment2：The effects of Different diets on milk production and milkcomposition of dairy cows
The second part was mainly conducted to study the effects of three diets on milkproduction and milk composition of dairy cows, and provide the data to the research ofmilk protein synthesis in the mammany gland. Thirty multiparous Holstein cows wereassigned to a single factor block design and cows were divided into three groups.Threediets were:1）Mixed forage represent Large-scale dairy farming.2) High-concentrate andcorn stover represent dairy farming.3) Low-concentrate and corn stover represent retaildairy farming.The exprement period were12weeks, and pre-feeding period were3weeks, every3weeks for a test cycle.Feed and milk sample were collected at15～18d ineach cycle.The results showed that: The MF group had a best performance, there was no difference between CSA and MF in milk protein yield and milk proten content, however,the Lactation efficiency was higher in MF vs.CSA. Compared to the CSA and MF, CSBhad lower level of all indicators of milk composition except milk fat. The results suggestthat, blance diets more efficient in nutrients metabolism, the energy level of CSB can notmaintain the lactating.
Experiment3: The effects of Different diets on metabolin content related to milkprotein synthesis in artery/vein blood of dairy cows
Based on experiment2, the objective of this part was to evaluate the metabolincontent related to milk protein synthesis in artery/vein blood of dairy cows and found outthe key factor that affect milk protein synthesis.The results showed that:PRL,GH,INR,IGF-1and glouse content in artery/vein blood have no difference indifferent diets(P>0.05), the level of BUN and leptin in EPA were higher in CSAvs.CSB and MF significantly, compare to the MF, CSA and CSB were significant higherin AST. Most of the amino acids have no difference in different diets, but, the ratio ofAsp, Arg, Thr, His, Gly and Leu have significant difference or difference in the trend.The results suggest that, CSA and CSB have low efficiency of nitrogen utilization. Leptinmay affect the synthesis of milk fat in the CSA group. The health of the liver wasaffented by CSA and CSB, perhaps.
Experiment4: The effects of Different diets on AA uptake and utilization inmamany gland of dairy cows
Based on experiment2, the objective of this part was to evaluate the difference ofAA uptake and utilization in mammary gland.The results showed that: the AA utilizationof mammary gland of CSB was restricted by the level of EAA in EPA, the reason was thelow level of nutrition and blood flow. There was no difference in AA uptake andutilization between CSA and MF.
Experiment5: Effects of different diets on the Gene Expression Related to MilkProtein Synthesis in the Mammary Gland of dairy cows
Based on experiment2, Mammary gland tissue (ca.500mg/animal) for RNAextraction were randomly performed for half numbers of cows in each group aftermilking on the last day of experiment. The expression of CSN1S1, CSN1S2,CSN3,INSR,PRLR, GHR1A, JAK2，STAT5and mTOR genes were determined by RT-QPCR method.The results showed that: Different diets affected the efficiency of milk protein synthesisin lactating Holstein cows. The balance dites can promote PRLR GHR1A and mTORgenes expression, resulting in increased milk protein synthesis.
The final results showed that: AA supply and utilization efficiency in mammarygland result in different milk protein and yield, milk protein synthesis efficiency wasevaluated by the gene expression related to milk protein synthesis in the mammary glandtissue. The milk protein AA pattern might increase major milk protein genes expression.Perhaps, the milk protein pattern can promote milk protein synthesis in mammary glandepithelial cell better.
引文
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