摘要
本研究目的在于揭示夏季热应激下补饲过瘤胃蛋氨酸对奶牛生产性能及淋巴细胞凋亡调控的影响。通过定期测定过瘤胃蛋氨酸对奶牛产奶量、乳成分的影响以及血液氨基酸变化趋势；全血自动分析仪分析奶牛外周血液学指标；流式细胞仪分析淋巴细胞凋亡率；采用夹心ELSIA的方法测定血清HSP70OD值；放射免疫方法测定奶牛血清T_3、T_4和皮质醇水平；半定量RT-PCR分析外周血液淋巴细胞HSF1mRNA、Bcl-2mRNA、BaxmRNA基因表达水平；荧光定量PCR分析Bcl-2mRNA、BaxmRNA、p53mRNA基因表达。研究结果如下：
1过瘤胃蛋氨酸对奶牛产奶量、乳成分以及血液氨基酸含量的影响
选用16头泌乳中期奶牛按拉丁方设计进行试验，研究饲粮中补充过瘤胃蛋氨酸0，8，12和16g．d~(-1)对产奶量、乳成分以及血液氨基酸含量的影响。结果表明，与对照组相比，补饲过瘤胃蛋氨酸能够提高奶牛乳蛋白、乳脂率和酪蛋白水平。补饲过瘤胃蛋氨酸有增加产奶量的趋势，能提高高产奶牛乳蛋白和乳脂肪量。9头泌乳中期荷斯坦奶牛在补饲过瘤胃蛋氨酸和普通蛋氨酸前后连续采血44小时，探索蛋氨酸的吸收变化曲线，发现补饲过瘤胃蛋氨酸后12～16小时达到吸收高峰，过瘤胃蛋氨酸能显著提高奶牛血清蛋氨酸水平，而补饲普通蛋氨酸则无明显影响。补饲过瘤胃蛋氨酸和普通蛋氨酸对血清总氨基酸水平均无显著影响。
16头泌乳奶牛进行配对试验，研究饲粮中补充12 g．d~(-1)过瘤胃蛋氨酸对热应激下奶牛产奶量和乳成分的影响。结果表明，与对照组相比，补饲过瘤胃蛋氨酸有促进产奶，提高奶牛乳蛋白、乳脂率和非脂固形物以及降低牛奶中体细胞数的趋势。表明奶牛补饲过瘤胃蛋氨酸有促进产奶，减缓热应激对奶牛泌乳的影响，提高奶牛血液中蛋氨酸的水平，但不能显著改变血液总氨基酸的水平。
2过瘤胃蛋氨酸对夏季泌乳奶牛外周血液指标的影响
全自动血细胞分析仪分析日平均温度在33℃下，奶牛补饲不同剂量的过瘤胃蛋氨酸，分析奶牛外周血液红细胞计数(red blood cell，RBC)、血红蛋白(hemoglobin，HGB)、白细胞总数(White blood cell，WBC)、中性粒细胞百分比(GRAN％)、淋巴细胞百分比(LYMPH％)、单核细胞百分比(MID％)、中性粒细胞计数(granulocyte，GRAN)、淋巴细胞计数(lymphocyte，LYMPH)、单核细胞计数(mid-cell，MID)、红细胞压积(hematocrit，HCT)、平均红细胞体积(mean corpuscular volume，MCV)、平均血红蛋白量(mean corpuscular hemoglobin，MCH)、平均血红蛋白浓度(mean corpuscular hemoglobin concentration，MCHC)、红细胞分布宽度(red cell distribution width，RDW)、血小板计数(platelet-count，PLT)、平均血小板体积(mean platelet volume，MPV)和血小板压积(platelet-crit，PCT)。16头泌乳奶牛进行配对试验，随机分为对照组和试验组，研究饲粮中补充12 g．d~(-1)过瘤胃蛋氨酸，分析血清中谷丙转氨酶(GPT)、谷草转氨酶(GOT)、总蛋白(TP)、白蛋白、球蛋白、葡萄糖(GLU)、乳酸脱氢酶(LDH)、碱性磷酸酶(ALP)、磷酸肌酸激酶(CPK)、超氧化物歧化酶(SOD)、谷胱苷肽过氧化物酶(GSH-Px)、和丙二醛(MDA)。
研究发现过瘤胃蛋氨酸能够改变奶牛外周血常规指标，饲喂过瘤胃蛋氨酸极显著的降低夏季奶牛外周血液红细胞计数(P<0.01)；血红蛋白呈增加趋势，20g．d~(-1)组差异极显著(P<0.01)，不同剂量过瘤胃蛋氨酸对血红蛋白影响差异不明显。饲喂过瘤胃蛋氨酸有提高奶牛外周血液中白细胞总数、中性粒细胞计数和淋巴细胞计数趋势；饲喂20 g．d~(-1)过瘤胃蛋氨酸能够明显提高奶牛外周血液单核细胞计数(P<0.05)。饲喂过瘤胃蛋氨酸，血液红细胞压积呈下降趋势，平均红细胞体积、平均血红蛋白量和平均红细胞蛋白浓度呈上升趋势，20 g．d~(-1)过瘤胃蛋氨酸显著提高红细胞分布宽度(P<0.05)；血小板计数呈上升趋势，其中16 g．d~(-1)和20 g．d~(-1)过瘤胃蛋氨酸能显著提高血小板计数(P<0.05)；平均血小板体积下降，血小板压积呈上升趋势。
与对照组相比，饲喂12 g．d~(-1)过瘤胃蛋氨酸，奶牛血液中谷丙转氨酶(GPT)、谷草转氨酶(GOT)、白蛋白水平和磷酸肌酸激酶(CPK)均呈下降趋势；补饲过瘤胃蛋氨酸能够明显降低奶牛外周血液中乳酸脱氢酶(LDH)水平(P<0.05)；总蛋白、球蛋白、碱性磷酸酶(ALP)和葡萄糖(GLU)水平呈增加趋势。与对照组相比，补饲过瘤胃蛋氨酸能够提高奶牛外周血液中SOD和GSH-Px水平，降低MDA水平。过瘤胃蛋氨酸能够促进奶牛血液中血红蛋白量和血小板等的生成，改变白细胞类型，表明过瘤胃蛋氨酸有提高降低奶牛热应激，促进细胞分化的作用，提高机体免疫力的作用。能够减弱热应激对奶牛肝脏和肌肉细胞的损伤，保护细胞膜免受热应激的损伤等作用，有缓解热应激和加速自由基的清除，减少自由基对细胞膜和DNA的损伤，提高抗氧化酶的活力。
3过瘤胃蛋氨酸对夏季泌乳奶牛外周血液淋巴细胞凋亡的影响
用流式细胞仪分析夏季热应激下奶牛补饲过瘤胃蛋氨酸对其外周血液淋巴细胞凋亡率的影响。结果发现对照组淋巴细胞凋亡率最高(8.38％)，饲喂过瘤胃蛋氨酸能够明显降低夏季高温奶牛外周血液淋巴细胞凋亡率，12 g．d~(-1)过瘤胃蛋氨酸组淋巴细胞凋亡率最低(2.43％)，16和20 g．d~(-1)过瘤胃蛋氨酸组淋巴细胞凋亡率分别为3.57％和3.92％，与对照组相比，差异极显著(P<0.01)。表明过瘤胃蛋氨酸能够抑制因高温热应激而导致的细胞凋亡，并与蛋氨酸的添加量有关。
4过瘤胃蛋氨酸对夏季泌乳奶牛血清HSP70和T_3、T_4以及皮质醇的影响
采用夹心ELSIA的方法测定血清HSP70OD值，放射免疫方法测定奶牛血清T_3、T_4和皮质醇水平，研究补饲过瘤胃蛋氨酸对奶牛血清HSP70、T_3、T_4以及皮质醇分泌的影响。结果发现，与对照组相比，饲喂12和16 g．d~(-1)过瘤胃蛋氨酸组，奶牛血清中HSP70O值分别提高了52.61％和51.51％，差异显著(P<0.05)，饲喂20 g．d~(-1)过瘤胃蛋氨酸组，奶牛血清中HSP70 OD值提高了26.47％，差异不显著(P>0.05)。与对照组相比，饲喂12、16和20 g．d~(-1)过瘤胃蛋氨酸，奶牛血清T_3浓度分别提高了6.94％、5.57％和5.26％(P>0.05)；奶牛血清T_4浓度分别提高了26.19％、26.83％和33.88％(P>0.05)。12 g．d~(-1)组奶牛血清皮质醇水平下降了12.15％，16和20 g．d~(-1)组奶牛血清皮质醇水平分别提高了42.99％和78.26％(P>0.05)。表明夏季奶牛补饲过瘤胃蛋氨酸，能够促进HSP70的表达，缓解热应激对奶牛血液皮质激素分泌的影响。
5过瘤胃蛋氨酸对奶牛外周血液淋巴细胞凋亡相关基因表达的影响
用半定量RT-PCR分析过瘤胃蛋氨酸对奶牛外周血液淋巴细胞HSF1mRNA、Bcl-2mRNA、BaxmRNA基因表达水平的影响。结果发现，补饲12 g．d~(-1)过瘤胃蛋氨酸组奶牛外周血液淋巴细胞HSF1mRNA水平最低，与对照组相比差异显著(P<0.05)，与对照组相比，补饲16和20 g．d~(-1)过瘤胃蛋氨酸组奶牛外周血液淋巴细胞HSF1mRNA丰度降低，但差异不显著(P>0.05)；补饲过瘤胃蛋氨酸能够提高奶牛外周血液淋巴细胞Bcl-2mRNA丰度，在12 g．d~(-1)组Bcl-2mRNA丰度最高，与对照组和16 g．d~(-1)组相比，差异极显著(P<0.01)，与20 g．d~(-1)组相比，差异不显著，16 g．d~(-1)和20 g．d~(-1)组与对照组相比，差异也不显著(P>0.05)；补饲过瘤胃蛋氨酸能够降低夏季奶牛外周血液淋巴细胞Bax-αmRNA丰度的降低，12 g．d~(-1)组最低，但差异均不显著(P>0.05)；Bcl-2mRNA/Bax-αmRNA在12 g．d~(-1)过瘤胃蛋氨酸组最高，与对照组相比，差异极显著(P<0.01)，16和20 g．d~(-1)与对照组相比，差异不显著(P>0.05)。表明过瘤胃蛋氨酸能够抑制奶牛淋巴细胞Bax和HSF1基因的表达，促进Bcl-2基因表达，抑制淋巴细胞凋亡。
选用16头泌乳奶牛，试验组8头补充12 g．d~(-1)过瘤胃蛋氨酸，用荧光定量PCR法研究对夏季奶牛外周血液淋巴细胞凋亡基因的影响。结果表明，饲喂12g．d~(-1)过瘤胃蛋氨酸组奶牛在第7d外周血液淋巴细胞Bcl-2 mRNA基因丰度提高了14.12％，BaxmRNA基因丰度下降了35.05％，Bcl-2mRNA与BaxmRNA比值提高了92.15％，但差异均不显著(P>0.05)；p53mRNA基因丰度降低了40.4％，差异极显著(P<0.01)。与对照组相比，在第14d过瘤胃蛋氨酸组奶牛外周血液淋巴细胞Bcl-2 mRNA基因丰度提高了27.86％，BaxmRNA基因丰度下降了18.86％，Bcl-2mRNA与BaxmRNA比值提高了67.51％，但差异均不显著(P>0.05)；p53mRNA基因丰度降低了49.53％，差异极显著(P<0.01)。表明夏季奶牛补饲过瘤胃蛋氨酸，能够抑制促凋亡基因的表达，上调抑凋亡基因的表达水平，保护淋巴细胞免受损伤。
1 The effects of rumen protected Methionine on performances and milk composition and amino acids of plasma in dairy cows
16 Holstein dairy cows in their midlactation were selected for a 4×4 Latin-square design study, to investigate the influence of a rumen protected methionine(RP-Met) source at dosage of 0, 8, 12 and 16g.d~(-1) on Met absorption, milk yield, milk protein and fat. The results showed that the supplementation with rumen-protected methionine increased milk protein and fat and casein. The supplementation with rumen-protected methionine had the tendency to increase of milk yield. The quantity of milk protein and fat were improved by rumen-protected methionine in high yielding cows. 9 Holstein dairy cows in their midlactation were taken samples for 44h before and after supplementing methionine. The result found that the addition of Smartamine led to a sharp increase in the level of serum Met with a peak from 12 to 16 h following the intake. The supplementation with rumen-protected methionine increased the level of serum Met significantly, and the addition of DL-Met did not achieve such response. The supplementationwith rumen-protected methionine and DL-Met had no significant effect on serum total amino acids.
16 cows were selected to study the effects of 12 g.d~(-1) rumen protected Methionine on the milk yield and composition in heat shocked condition. The results showed that supplementing rumen protected Methionine had the tendency to improve milk protein and fat, to decrease SCC. The supplementation with rumen-protected Methionine could improve milk yield and Methionine of plasma and decrease heat shock status in dairy cows. But it would not improve to the level of lysine and total amino acids.
2 The effects of rumen protected Methionine on the blood indexes in peripheral blood of dairy cows
Supplementing the different dosages(0,12,16 and 20 g.d~(-1)) with rumen protected Methionine in order to analyze the blood indexes by automatic analyzer of blood. The blood indexes included as follows: white blood cell(WBC), lymphocyte(LYMPH), granulocyte(GRAN), mid-cell(MID), LYMPH%, MID%, GRAN%, red blood cell(RBC), hemoglobin(HGB), mean corpuscular volume(MCV), hematocrit(HCT), mean corpuscular hemoglobin(MCH), mean corpuscular hemoglobin concentration(MCHC), red cell distribution width(RDW), platelet count(PLT), mean platelet volume(MPV) and platelet-crit(PCT). 16 cows were selected to study the effects of 12 g.d~(-1) rumen protected Methionine on blood indexes in heat shocked condition. The blood indexes included as follows: GPT, GOT, TP, Albumin, Globin, GLU,LDH, ALP, CPK, SOD, GSH-Px, MDA. The results were showed as follows: Supplementing rumen protected Methionine could significantly decrease RBC(P<0.01). 20 g.d~(-1) rumen protected Methionine could significantly HGB, and HGB had the increased tendency with the dosages. WBC and LYMPH and GRAN had the tendency to increase with supplementing rumen protected Methionine. It is significant to the dosage of 20 g.d~(-1). HCT had the decreasing tendency with supplementing rumen protected Methionine. MCV and MCH and MCHC had the increasing tendency with supplementing rumen protected Methionine. RDW improved significantly in the dosage of 20 g.d~(-1). The dosages of 16 g.d~(-1) and 20 g.d~(-1) could significantly improve PLT. MPV had the decreased tendency and PCT improved. GPT and GOT and albumin and CPK had the decreased tendency with the supplement with rumen protected Methionine. LDH had been improved significantly if supplemented rumen protected Methionine(P<0.05). Total album and globulin and ALP and GLU had the improved tendency to supplement rumen protected Methionine. Supplementing rumen protected Methionine could improve SOD and GSH-Px and decrease MDA in peripheral blood of dairy cows. The supplementation of rumen protected Methionine had the tendency to improve HGB and PLT. The types of WBC were transferred. The supplementation of rumen protected Methionine had the role on decreasing the damage of cell in heat shocked dairy cows.
3 The effects of rumen protected Methionine on apoptosis of lymphocyte in heat shocked dairy cows
40cows were selected in the mid-lactation to study the effects of 0,12,16 and 20 g.d~(-1) rumen protected Methionine on the apoptotic rate of blood lymphocyte. The apoptotic rate of lymphocytes was analyzed by flow-cytometry. The results found that the apoptotic rate of control was 8.38%. It is the highest. Supplementing rumen protected Methionine reduced significantly the apoptotic rate of lymphocytes in summer. The apoptotic rate of 12,16 and 20 g.d~(-1) reduced respectively 71%, 57.4% and 53.22% than that of control(P<0.01).The supplementation of rumen protected Methionine could control the apoptosis of lymphocytes because of heat stress.
4 The effects of rumen protected Methionine on HSP70 OD and T_3 and T_4 and cortisol in the heat shocked dairy cows
Double antibodies sandwich ELISA method for detecting HSP70 in plasma and RIA method for detecting T_3 and T_4 and cortisol. We studied the effects of rumen protected Methionine on of HSP70 ODand T_3 and T_4 and cortisol in the heat shocked dairy cows. The results showed HSP70OD improved respectively 52.61% and 51.51% and 26.47% in the treatments than that of control. T_3 and T_4 of plasma had the increased tendency in the treatments than that of control. Cortisol of plasma in the dosage of 12 g.d~(-1) decreased 12.15% than that of control. But cortisol of plasma improved respectively 42.99% and 78.26% in the dosages of 16 and 20 g.d~(-1) than that of control. It is not significant. The supplementation of rumen protected Methionine improved metabolism and decreased the heat stress of dairy cows.
5 The effects of rumen protected Methionine on expression of related gene for apoptosis of lymphocyte in dairy cows
The gene effects of HSF1mRNA and Bcl-2mRNA and BaxmRNA were analyzed by RT-PCR in the blood lymphocytes of dairy cows. The results showed that supplementing rumen protected Methionine could reduce the abundance of HSF1mRNA than that of control. Supplementing rumen protected Mthionine improved the abundance of Bcl-2mRNA than that of control. It is highest in the treatment of 12 g.d~(-1). Supplementing rumen protected Mthionine reduced the abundance of BaxmRNA than that of control. It is lowest in treatment of 12 g.d~(-1). Supplementing rumen protected Methionine improved Bcl-2mRNA/Bax-amRNA.
16 cows were selected to study the effects of rumen protected Methionine on expression for apoptotic gene in heat shocked dairy cows. Bcl-2mRNA and BaxmRNA and p53mRNA were analyzed by quantificational PCR. The results showed that the abundance of Bcl-2mRNA improved 14.12% than that of control in seventh day. The abundance of BaxmRNA decreased 35.05% than that of control in seventh day. The ratio of Bcl-2mRNA/BaxmRNA was improved 92.15% than that of control(P>0.05). p53mRNA decreased 40.4% than that of control in seventh day(P<0.01). The abundance of Bcl-2mRNA improved 27.86% than that of control in 14th day. The abundance of BaxmRNA decreased 18.86% than that of control in 14th day. The ratio of Bcl-2mRNA/BaxmRNA was improved 36.36% than that of control(P>0.05). p53mRNA decreased 49.53% than that of control in 14th day(P<0.01). Supplementing rumen protected Methionine had the role on improving genic expression of Bcl-2 and controlling genic expression of Bax and p53.
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