【作者】 张健；

【导师】 邵涛； 蒋永清；

【作者基本信息】 南京农业大学 ， 动物营养与饲料科学， 2009， 硕士

【摘要】 热应激对奶牛的生产性能和繁殖性能有极大的负面影响。本研究从营养和代谢调控两方面探索缓解奶牛的热应激的途径,同时利用RAPD技术筛选奶牛的耐热性分子标记。1.试验一饲喂高能饲料对热应激条件下奶牛的生产性能和生理生化指标的影响及其机理的探究。选择33头处于热应激期泌乳中期的荷斯坦奶牛,随机分为对照组和处理Ⅰ、Ⅱ。处理Ⅰ、Ⅱ饲喂不同的高能饲料,分别为烘烤大豆和油菜籽,对照组为常规饲料。结果表明：处理Ⅰ试验期的产奶量比对照组高3.27%(P<0.05),处理Ⅰ、Ⅱ试验期产奶量比试验前分别提高了2.21%和7.31%,而对照组则略有下降；不同处理间的直肠温度相近,但处理Ⅰ组呼吸频率极显著高于对照组(P<0.01),两处理组的心跳次数显著高于对照组(P<0.05)。与对照组相比,处理组Ⅰ和Ⅱ的过氧化氢酶(CAT)、丙二醛(MDA)和K+的含量显著提高(P<0.05),血清总蛋白(TP)、Cl-含量显著降低(P<0.05)。处理组Ⅰ的血清尿素氮(BUN)含量显著高于对照组(P<0.05)；处理组Ⅱ的皮质醇(Cor)显著高于对照组(P<0.05)。处理组Ⅰ和Ⅱ的T3、白细胞介素(IL-2)、免疫球蛋白(IgG)、超氧化物岐化酶(SOD)和胰岛素(INS)的分泌量有增加的趋势(P>0.05),而催乳激素(PRL)、肌酸激酶(CK)和Na+分泌量有下降的趋势(P>0.05)。处理组Ⅰ的T4和乳酸脱氢酶(LDH)的含量有升高的趋势(P>0.05),而处理组Ⅱ呈降低的趋势(P>0.05)。2.试验二添加复合添加剂对热应激条件下奶牛的生产性能和生理生化指标的影响及其作用机理。选择22头处于泌乳中期的荷斯坦奶牛,随机分为对照组和处理组。处理组添加复合添加剂,对照组为常规日粮。结果表明：使用复合添加剂饲料后,处理组试验期的产奶量比对照组提高1.52%(P<0.05),对照组试验期产奶量比试验前下降16.36%,而试验组提高了1.45%；使用复合添加剂除乳脂率有明显的降低外(P<0.05),对其它乳成分基本没有影响；与对照组比较,试验组的直肠温度显著降低(P<0.05),呼吸频率显著升高(P<0.05)。与对照组比较,处理组的T3、Cor、INS、IL-2、TP、CK、SOD和LgG都有升高的趋势(P>0.05),而T4和Cl-显著升高(P<0.05)。处理组和对照组相比,PRL、LDH、MDA、K+和Na+的含量都有下降的趋势(P>0.05)；而CAT显著下降(P<0.05)。BUN的含量两处理组间没有明显变化(P>0.05)。3.试验三用RAPD技术筛选奶牛的耐热性分子标记。以日均产奶量为标准从处于热应激期的52头奶牛中选出22头奶牛(12头为耐热性优良奶牛,10头为非耐热奶牛)为试验动物,通过RAPD技术进行耐热分子标记的筛选。研究不同酶和反应循环数对RAPD反应的影响,建立了一套适合本试验最佳的反应体系。对170条S系列随机引物进行了PCR-RAPD扩增反应,具有多态性的引物26条,筛选出稳定性好、带型清晰的引物3条。结合奶牛的生理生化指标,发现有两条引物S275和S2011的扩增效果与某些生理生化指标存在显著相关：S275和$2011的差异片段与产奶量(P<0.01)、T4(P<0.05)呈负相关,与乳脂率呈中度的正相关(P<0.05),其中S275的差异片段还与乳蛋白率(P<0.05)、CK(P<0.05)显著正相关,与钾离子含量呈显著负相关(P<0.05),S2011的差异片段与钠离子含量显著负相关(P<0.05)。对两条引物特异性片段测序结果的BLAST分析发现,引物S2011特异性条带在牛第4号染色体上二肽基肽酶Ⅵ下游32Kb处；引物S275测序结果有两个序列,其中序列Ⅰ在MHCⅠ类样基因家族A1基因下游的0.7Kp,ULBP27上游和下游的1.6Kb等基因调控区附近均有分布,而序列Ⅱ位于牛的3号染色体上的NOL1/NOP2/Sun domain family, member 4.通过BLAST结果可以初步推测引物S2011和S275可以作为筛选奶牛耐热性状的分子标记。本研究通过添加烘烤大豆和油菜籽研究高能饲料对奶牛生产性能和生理生化指标的影响,探讨一种用于缓解奶牛热应激的新型能量饲料的机理：论文还从多种抗热应激添加剂复合效果方面入手,进一步探讨缓解奶牛热应激的新途径；同时利用RAPD技术筛选出与耐热性能有关的分子标记,并结合奶牛的生理生化指标进行分析,确定了其可行性。更多还原

【Abstract】 Heat-stress has a huge negative effect on production and reproduction performance of dairy cattle. Explore to ease the heat-stress through the aspects of nutrition and metabolism, while RAPD technology was used to select heat resistance molecular markers on dairy cattle.1.The experiment 1 was conducted to study the effects and mechanism of high energy feed on biochemical physiologic indices and production performance under heat-stress. Thirty three lactating cows were used in the experiment and divided into three groups randomly with 11 cows in each group. The cows in control group were fed with basic diets, the treatmentⅠwas supplemented with roast soybean, and treatmentⅡwas supplemented with rapeseed. The results showed that the milk yield of treatmentⅠhad significant (P<0.05) increase of 3.27% compared with control, milk yield of treatmentⅠandⅡboth increased by 2.21% and 7.31% respectively compared with previous. However, the control group had slight declining. The breath frequency of treatmentfⅠhad significantly (P<0.05) increased compared with that of the control group, and the two treatments had significant higher heartbeat times (P<0.05) compared with control.Both of treatedⅠandⅡhad an significant (P<0.05) increase in the contents of CAT,MDA and K+, but an significant decrease in contents of TP and Cl- compare with the control. TreatedⅠsignificantly(P<0.05)increased the BUN content. Cortical increased significantly (P<0.05) in the treatmentⅡas compared with control.2.The experiment 2 was conducted to study the effect of complex additive on milk performance and biochemical physiology parameter of cows under heat-stress and its mechanism. Twenty two lactating cows were divided randomly two groups with 11 cows in every group. The cows in control group were fed with basic diets; the treatment was supplemented with complex additive. The results showed that the milk yield of control had decreased 16.36% compare with before experiment, however, that of treatment had increased 1.45%. The complex additive had no effect on the milk composition, except fat content with significant decrease(P<0.05); The recta temperature of treatment had significantly (P<0.05) decreased,and the breath frequency had significantly (P<0.05) increased compare with control. Compared with the control,T3、Cor、INS、IL-2、TP、CK、SOD and LgG trended to increase(P>0.05), however, the content of T4 and cl- had an significant (P<0.05) increase. Compared with the control group, the content of PRL、LDH、MDA、K+ and Na+ trended to decrease (P>0.05), but the content of CAT had an significant (P<0.05) decrease.3.In this study,22 cows were selected from 52 cows based on average daily milk yield under heat-stress. Then select dairy heat-resistance molecular markers using RAPD. The effects of different enzyme and cycles on RAPD were analyzed. An optimization RAPD system was foud.26 primers with better effect were selected in 170 random primers using PCR-RAPD.Considered the detected result of biochemical and physiology parameters, two molecular markers relative to heat-tolerance in Holstein Cattle were selected finally. There were significantly positive correlations (P<0.05)between milk fat percentage and different fragments of primer S275 and S2011; the content of K+ and milk protein rate (P<0.05)with different fragment of primer S275 was a significantly negative correlations;T3、T4 (P<0.05)and milk yield (P<0.01)with different fragment of primer S275 and S2011 were a significant negative correlations; the content of CK with different fragment of primer S275 was a significantly positive correlations(P<0.05);There were a significantly negative correlations (P<0.05)between Na+ and different fragment of primer S2011.Analysis of sequencing results of specific fragment using BLAST, there were specific bands of primer S2011 at Dipeptidy-peptidase 6 downstream of 32Kb of Bos taurus chromosome 4 genomic. There were two Sequence in sequencing results of primer S275, one sequence was on MHC Class I-like Family Al downstream of 0.7Kb, and ULBP27 upstream of 10Kb; however, another sequence on NOL1/NOP2/Sun Domain Family of Bos taurus chromosome 4 genomic. It was suggested that primers S2011 and S275 could be used as molecular markers in filtration heat-resistant traits of dairy cattle from the result of BLAST.This study was conducted to study the effects and mechanism of high energy feed and complex additive on biochemical physiologic indices and production performance under heat-stress. It was suggested that two molecular markers were screened in heat-resistant using RAPD technology combined with physiological and biochemical indicators of dairy.更多还原