植物源性物质对生长湖羊瘤胃发酵和肉品质的影响及其作用机理的研究
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摘要
植物源性物质由于含有多种具有抗菌、抗氧化等生物活性成分而越来越受到动物营养学家的关注。本研究以皂甙类物质茶皂素、富含不饱和脂肪酸的豆油和含有黄酮类和挥发油类物质的杭白菊茎叶等植物源性物质为材料,分三部分六个试验研究了其对湖羊断奶羔羊胴体品质、瘤胃发酵以及肌肉脂肪酸组成的影响,并利用荧光定量PCR等分子生物学技术,研究瘤胃微生物数量的变化以及与肌肉脂肪酸合成相关酶基因mRNA丰度的变化,揭示植物源性物质影响湖羊肌肉脂肪酸组成的机理。
第一部分植物源性物质对生长湖羊胴体品质的影响(试验一和试验二)
试验一研究了日粮中添加茶皂素和豆油对生长湖羊血清生化指标和胴体品质的影响。选择健康湖羊断奶羔羊32只(14.2+1.38kg),随机分为四个日粮处理组：不加茶皂素也不加豆油组(NTNS);茶皂素组(TS)；豆油组(SO)；茶皂素和豆油混合添加组(TS-SO).试验采用二因素随机试验设计。茶皂素的添加量为每只羊3g／d,豆油的添加量为日粮干物质的3%。动物接近自由采食,但在组间控制采食量,以减少因采食和增重差异造成的肉质差异。饲养试验结束后,将羊放入呼吸代谢室进行甲烷测定。甲烷排放测定结束后,所有动物颈静脉采血,用于测定血清生化指标。然后每组取4组羊进行屠宰试验。结果发现,TS和SO的添加可显著降低甲烷的产量(P<0.05),与NTNS组相比,TS,SO和TS-SO组每千克干物质采食量所产生的甲烷量分别降低了27.7%,18.9%和13.9%。添加TS对羔羊血清生化指标不产生显著影响,但是SO和TS-SO组的总蛋白,高密度脂蛋白胆固醇,胆固醇和游离脂肪酸的含量显著高于NTNS组,葡萄糖的含量则显著低于NTNS组(P<0.05)。SO组的低密度脂蛋白胆固醇含量显著高于TS组(P<0.05)。各组间胴体品质没有显著差异(P>0.05)。这些结果表明：添加TS和SO可显著降低甲烷排放,减少饲料能的损失,但对羔羊胴体品质没有影响,SO的添加可影响羔羊血清生化指标。
试验二研究了杭白菊茎叶对湖羊断奶羔羊生长性能、血清生化指标和胴体品质的影响。试验选取健康断奶湖羊羔羊36只(16+2.6kg),依据随机区组设计和同栏大小相近原则,根据体重分成4个处理,每个处理3个重复(栏),每个栏3只羔羊。试验采用精粗比为30：70的全混合颗粒饲粮,四组粗料中稻草和杭白菊茎叶(CSL)的比例分别为70:0 (CSLO),45:25 (CSL25),25:45 (CSL45)和0：70(CSL70)。试验结果,随着CSL饲喂比例的增加,羔羊的平均日增重增加,血清尿素氮(BUN)含量提高,CSL0.组和CSL70组间差异达显著水平(P<0.05)。CSL对羔羊血清过氧化氢酶(CAT)、谷胱甘肽过氧化物酶(GSH-Px)活力、总抗氧化能力(T-AOC)和血清丙二醛(MDA)含量均有不同程度的影响。随着CSL饲喂比例增加,羔羊血清中CAT和T-AOC增加,CSL45组和CSL70组与CSLO组相比,差异显著(P<0.05)；CSL45组GSH-Px活力显著高于其余各组(P<0.05)；MDA随CSL饲喂比例的增加而减少,CSLO和CSL70组间差异显著(P<0.05)。CSL的饲喂对羔羊胴体品质不存在显著影响,但是屠宰率CSL70组显著高于CSLO组(P<0.05)。上述结果表明,CSL用作粗料饲喂断奶湖羊可以提高抗氧化能力,促进生长。
第二部分植物源性物质对生长湖羊瘤胃发酵的影响(试验三和试验四)
试验三研究茶皂素和豆油对羔羊瘤胃发酵参数以及瘤胃微生物区系的影响。待饲养试验结束后,动物屠宰后取瘤胃液样品,测定瘤胃发酵参数及瘤胃微生物区系。结果显示：与NTNS组相比,TS,SO和TS-SO组的瘤胃pH值均显著降低(P<0.05)。添加TS和SO显著提高总挥发性脂肪酸(VFA)含量(P<0.05),但是对单独的VFA不存在显著影响(P>0.05)。氨态氮的含量在SO和TS-SO组显著低于NTNS组(P<0.05),但是TS组变化不大。与NTNS组相比,TS,SO和TS-SO组的微生物蛋白的含量均显著提高。添加TS和SO对真菌数量没有显著影响(P>0.05),TS作为主效应对原虫数量影响不显著,但是通过二次分析发现,TS对原虫的作用取决于SO,单独添加效果是显著的(P=0.04),但是与SO同时添加效果则不显著(P=0.68)。添加SO显著降低甲烷菌的数量(P<0.05)。SO的添加显著降低黄色瘤胃球菌和产琥珀酸丝装杆菌的数量(P<0.05),但是TS对这两个纤维菌没有影响。从DGGE图谱来看,日粮中添加TS和SO对瘤胃细菌、原虫以及甲烷菌区系有一定的影响,但是各组间条带数、多样性指数不存在显著差异(P>0.05)。结论：添加茶皂素和豆油对羔羊瘤胃微生物数量和微生态区系均有一定影响。
试验四研究杭白菊茎叶对湖羊瘤胃发酵参数以及氢化相关微生物的影响。瘤胃液取自试验二。试验结果显示：CSL70组湖羊瘤胃氨态氮的含量显著高于其余各组(P<0.05),微生物蛋白的含量CSL45组和CSL70组显著高于CSLO组和CSL25组(P<0.05)。瘤胃液相中,CSL的饲喂降低瘤胃原虫的数量,且在CSLO组和CSL70组间差异达显著(P<0.05)；溶纤维丁酸弧菌的数量CSL25组显著高于CSLO组和CSL70组(P<0.05)；蛋白溶解梭菌的数量CSL45组显著高于CSLO组(P<0.05)。瘤胃固相中,瘤胃原虫的数量也随着CSL的饲喂而减少,CSLO组和CSL45组间差异显著(P<0.05)；溶纤维丁酸弧菌的数量CSLO组显著高于CSL70组(P<0.05),但是蛋白溶解梭菌没有显著的变化。结果表明,杭白菊茎叶可影响瘤胃氨态氮和微生物蛋白的含量,以及瘤胃氢化菌的生长,这些影响可能与其所含有的黄酮类和挥发油类物质相关。
第三部分植物源性物质对生长湖羊肌肉脂肪酸组成及相关酶基因表达的影响(试验五和试验六)
试验五研究茶皂素和豆油对羔羊肌肉脂肪酸组成以及相关酶基因表达的影响。肝脏和肌肉样品取自试验一。试验结果显示：与NTNS组相比,各处理组肌肉组织中饱和脂肪酸(SFA)含量均有所降低,其中TS组和TS-SO组的C14：0与C15：0达显著水平(P<0.05)。各处理组肌肉组织中不饱和脂肪酸(UFA)的含量均有所增加,t11C18:1显著高于NTNS组(P<0.05)；SO组的cis-9, trans-11共轭亚油酸(CLA)显著高于其余各组(P<0.05)；SO组的多不饱和脂肪酸(PUFA)高于NTNS组(P<0.05)。日粮中添加TS和SO对羔羊肝脏中脂蛋白酯酶(LPL)基因的mRNA丰度不存在显著影响(P>0.05),但与NTNS组相比,TS和SO组肝脏组织中硬脂酰辅酶A去饱和酶(SCD)基因的mRNA丰度降低(P<0.05)。与NTNS组相比,TS和SO组肌肉组织中LPL基因mRNA丰度显著上升(P<0.05),且显著高于TS-SO组。肌肉组织中SCD基因mRNA丰度在SO组的表达显著高于其余各组(P<0.05)。因此推测添加SO对肌肉组织cis-9, trans-11CLA的影响受到SCD基因的调控。
试验六研究杭白菊茎叶对羔羊皮下脂肪和肌肉脂肪酸组成以及乙酰辅酶A羧化酶(ACC)基因在肝脏、皮下脂肪和肌肉组织中表达的影响。肝脏、皮下脂肪和肌肉样品取自试验二。结果显示：饲喂CSL可以提高羔羊皮下脂肪C16：1的含量,其中与CSLO组相比,CSL25组达显著水平(P<0.05)。C18：2的含量在CSL70组含量最高,显著高于CSLO组和CSL45组。总饱和脂肪酸和总不饱和脂肪酸随着CSL饲喂比例的增加而降低和增加,其中在CSLO组和CSL70组间差异达显著水平(P<0.05)。而饲喂CSL对羔羊肌肉中总不饱和脂肪和总饱和脂肪酸均没有显著影响(P>0.05)。CSL对肝脏、皮下脂肪和肌肉组织中ACC基因的表达均不存在显著影响(P>0.05)。ACC基因在肝脏中的表达量较少,比在脂肪和肌肉组织中表达量小2个数量级。结果表明：杭白菊茎叶的饲喂可影响动物脂肪和肌肉组织中脂肪酸的组成,但对ACC基因的表达没有影响。
综上所述,茶皂素和豆油的添加可以降低湖羊羔羊甲烷排放,从而减少饲料能的损失。日粮中添加茶皂素可能通过降低原虫数量从而降低羔羊肌肉组织中CLA的含量。添加豆油可降低肌肉组织中饱和脂肪酸,提高不饱和脂肪酸和CLA的含量,豆油对肌肉组织中cis-9, trans-11 CLA的合成可能是通过SCD基因调控实现。杭白菊茎叶饲喂羔羊可提高抗氧化能力,促进生长,且可通过杭白菊茎叶中的黄酮成分作用于瘤胃氢化菌而影响脂肪和肌肉组中的脂肪酸的组成,降低羔羊皮下脂肪组织中总饱和脂肪酸,增加不饱和脂肪酸含量,及影响肌肉组织中C18：0和C18：2的含量。
The components of plant orgin have many active properties, such as antibacterial and antioxidant. Thus, they couled be used in animal feed to replace the antibiotic. This study contained three parts including six experiments and it was conducted to investigate the effect of tea saponins (TS), soybean oil (SO) and chrysanthemun stem-leaf (CSL) on carcass quality, rumen fermentation and fatty acid profiles of muscle in growing lambs and approach to the mechanism involved.
Part I:Effect of components of plant origin on carcass quality of growing lambs. (Expt.1 and 2)
Experiment 1 was carried out to investigate the effects of TS, SO, and TS-SO on serum parameters and carcass quality of growing lambs. Thirty-two Huzhou lambs weaned at the age of 50 days, with an initial body weight of 14.2±1.38 kg, were assigned to four dietary treatments in a 2×2 factorial arrangement with TS (0 or 3g/d) and SO (0 or 3% of DM). The diet without additives was considered as NTNS (no TS or SO). Feed intake was controlled to be similar among the four groups to minimize the meat differences resultant from growth rate. After the feeding trial, four lambs from each treatment were moved to simple open-circuit respiratory chambers (two animals per chamber) to measure methane production. At the end of methane measurement, blood samples of all animals were taken via jugular vein puncture and serums were separated from the blood samples for analysis. Then animals were slaughtered and carcasses were weighed and dissected. No differences were observed in animal growth performances, carcass qualities among four treatments (P> 0.05). Addition of tea saponins had no effect on the serum parameters. Compared with NTNS, the contents of serum total protein, high density lipoprotein-cholesterol, cholesterol and non-esterified fatty acid were higher but the concentration of glucose was lower in SO and TS-SO groups than in NTNS group (P< 0.05). The content of low density lipoprotein-cholesterol was higher in SO group than in TS group (P< 0.05). These results indicated that addition of TS and SO can suppress the methane emission to improve the efficiency of feedstuff utilization, but have little effect on the carcass qualities of lambs.
Experiment 2 was conducted to determine the effect of feeding CSL on growth performance, serum parameters and carcass quality of growing lambs. Thirty-six Huzhou growing lambs were assigned to four groups of nine lambs each, and allotted to three units of three lambs at similar weight. The ratio of roughage to concentrate in all the diets was 70:30. Proportion of rice straw to CSL in roughage was 70:0 (CSLO),45:25 (CSL25),25:45 (CSL45), and 0:70 (CSL70) in diets, respectively. The trial lasted for 72 days with the first 12 days for adaptation. Feed ingested and refused were recorded every week, and body weight gain was measured every two weeks. At the end of the trial, serum biochemical and antioxidant parameters and carcass quality were determined. With increasing levels of CSL, average daily gain and serum urea nitrogen increased, with a significant difference between diets CSLO and CSL70 (P< 0.05). Serum catalase and total antioxidant capacity were significantly higher (P <0.05) in lambs on diets CSL45 and CSL70 than those on diet CSLO. The level of serum glutathione peroxidase was highest in animals on diet CSL45 (P< 0.05). Compared to that in diet CSLO, serum malondialdehyde was decreased in diet CSL70 (P< 0.05). Chrysanthemum stem-leaf had no effect on the carcass quality, but the dressing percent was higher in group CSL70 than in group CSLO (P< 0.05). From the present study, it is inferred that inclusion of CSL in the diets could increase antioxidant ability in growing lambs and improve growth performance.
Part II:Effect of components of plant origin on rumen fermentation and microbial populations in the rumen of lambs. (Expt.3 amd 4)
In Experiment 3, rumen fluids were sampled from the sheep used in Expt. 1 to determine the effects of addition of TS and SO on rumen fermentation and microbial populations in the rumen of growing lambs. The microbial communities were analyzed by real-time PCR and denaturing gradient gel electrophoresis technique. Populations of rumen methanogens, protozoa, fungi, Ruminococcus flavefaciens, and Fibrobacter succinogenes were expressed as a proportion of total rumen bacterial 16S rDNA. Ruminal pH was decreased (P< 0.05) for lambs fed diets with TS, SO, and TS-SO, vs. the NTNS, and ammonia N concentration was reduced by SO (P< 0.05). Concentrations of total volatile fatty acids were increased in all groups (P< 0.05), with no significant differences in proportions of individual acids among diets. Microbial protein was increased (P< 0.05) with TS, SO, and TS-SO. Addition of TS, SO, and TS-SO had little effect on fungal population (P> 0.05), but protozoa populations relative to total bacterial 16S rDNA were decreased (P< 0.05) for lambs fed diets with TS, SO, and TS-SO, with the lowest value in lambs fed the diet containing SO only. Population of methanogens was inhibited by SO (P< 0.05), but not by TS. Addition of SO and TS-SO had an inhibitory effect on the population of fibrolytic microbes including R. flavefaciens and F. succinogenes. Addition of TS and SO has an effect on the communities of bacterial, protozoa and methanogens. However, the diversities of bacterial, protozoa and methanogens were not affected by TS and SO. From the present study, it is inferred that the rumen microbial communities could be affected by addition of tea saponin and soybean oil.
Experiment 4 was conducted to determine the effect of feeding CSL on rumen fermentation and microbial populations in the rumen of growing lambs. The rumen samples both liquid and solid were taken from the animals in experiment 2. Ammonia N concentration was lowest in CSL70 (P< 0.05) and microbial protein was higher in CSL45 and CSL70 than in CSL0 and CSL25 (P < 0.05). In the liquid of rumen fluid, CSL decreased the protozoa populations; the population of B.fibrisolvens was higher in CSL25 than in'CSLO and CSL70 (P < 0.05); the C.proteoclasticum population was greater in diet CSL45 than in diet CSL0 (P< 0.05). In the solid of rumen fluid, the population of protozoa was decreased in diet CSL45 as compared to in the diet CSL0 (P< 0.05); the B.fibrisolvens population was larger in diet CSL0 than in diet CSL70 (P< 0.05); But the population of C.proteoclasticum was uninfluenced by CSL. It is inferred that the rumen fermentation and microbial populations could be affected by the flavone and naphtha components of CSL.
Part III:Effect of components of plant origin on fatty acid composition in longissimus dorsi muscle and expression of related enzyme gene. (Expt.5 and 6)
In Experiment 5, the longissimus dorsi (LD) muscle and liver tissuses were taken from the animals in Experiment 1 to determine the effects of addition of TS and SO on fatty acid profile in LD muscle, gene expression of lipoprotein lipase (LPL) and stearoyl-CoA desaturase (SCD) both in liver and muscle of growing lambs. Vaccenic acid percentage was enhanced (P< 0.05) in muscle of lambs fed TS and SO. The proportion of cis-9, trans-11 conjugated linoleic acid (CLA) was increased (P< 0.05) by SO, but decreased (P< 0.05) by addition of TS in LD muscle. The percentage of total saturated fatty acids (SFA) in muscle was decreased (P< 0.05) by addition of TS and SO, while addition of SO increased (P< 0.05) the percentage of total polyunsaturated fatty acids (PUFA). The ratio of cis-9, trans-11 CLA to vaccenic acid was decreased (P< 0.05) by TS, but increased (P< 0.05) by SO. Addition of TS and SO had no effect on the LPL gene expression in liver (P> 0.05). Compared with NTNS, the SCD mRNA expression in liver was increased in group TS and SO. The LPL mRNA expression in muscle was higher in group TS and SO than in NTNS. Addition of SO could increase the SCD mRNA expression (P< 0.05). These results indicated that inclusion of TS and SO in the diet of growing lambs affect the fatty acid profiles of LD muscle and that the proportion of cis-9, trans-11 CLA in the muscle influenced by TS and/or SO, may be regulated by SCD gene.
Experiment 6:This study was conducted to determine the effect of feeding CSL on fatty acid profile in LD muscle and subcutaneous fat, Acetyl CoA carboxylase (ACC) mRNA expression in liver, subcutaneous fat, and LD muscle. The liver, subcutaneous fat and LD muscle were taken from the animals in Experiment 2. In subcutaneous fat, the content of C 16:1 was increased by CSL; there was a highest proportion of C18:2 in diet CSL70 (P< 0.05); with increasing levels of CSL, the total SFA were decreased but the total undaturated fatty acids (UFA) were increased (P< 0.05). However, CSL did not affect the total SFA and total UFA in LD muscle. Inclusion of CSL had no effect on ACC mRNA expression in liver, subcutaneous fat, and LD muscle (P> 0.05). The ACC gene expression in liver was lower than in subcutaneous fat, and LD muscle. These results indicated that the fatty acid composition of subcutaneous fat and LD muscle could be affected by CSL, which may not be related to the ACC gene expression.
In summary, addition of TS and SO could reduce methane production to enhance the efficiency of feedstuff utilization. The intramuscular fat of lambs fed a diet containing TS had lower proportion of CLA. Dietary addition of linoleic acid-rich SO was effective in decreasing SFA proportion and increasing the proportion of PUFA and cis-9 trans-11 CLA in the LD muscle, which may be mediated through the expression of SCD gene. Inclusion of CSL in the diets could increase antioxidant ability in growing lambs and improve growth performance. The CSL could reduce the proportion of total SFA and increase the percentage of total UFA in subcutaneous fat, which may be related to the biohydrogenation associated bacteria in rumen affected by flavone in CSL,
第一部分植物源性物质对生长湖羊胴体品质的影响(试验一和试验二)
试验一研究了日粮中添加茶皂素和豆油对生长湖羊血清生化指标和胴体品质的影响。选择健康湖羊断奶羔羊32只(14.2+1.38kg),随机分为四个日粮处理组：不加茶皂素也不加豆油组(NTNS);茶皂素组(TS)；豆油组(SO)；茶皂素和豆油混合添加组(TS-SO).试验采用二因素随机试验设计。茶皂素的添加量为每只羊3g／d,豆油的添加量为日粮干物质的3%。动物接近自由采食,但在组间控制采食量,以减少因采食和增重差异造成的肉质差异。饲养试验结束后,将羊放入呼吸代谢室进行甲烷测定。甲烷排放测定结束后,所有动物颈静脉采血,用于测定血清生化指标。然后每组取4组羊进行屠宰试验。结果发现,TS和SO的添加可显著降低甲烷的产量(P<0.05),与NTNS组相比,TS,SO和TS-SO组每千克干物质采食量所产生的甲烷量分别降低了27.7%,18.9%和13.9%。添加TS对羔羊血清生化指标不产生显著影响,但是SO和TS-SO组的总蛋白,高密度脂蛋白胆固醇,胆固醇和游离脂肪酸的含量显著高于NTNS组,葡萄糖的含量则显著低于NTNS组(P<0.05)。SO组的低密度脂蛋白胆固醇含量显著高于TS组(P<0.05)。各组间胴体品质没有显著差异(P>0.05)。这些结果表明：添加TS和SO可显著降低甲烷排放,减少饲料能的损失,但对羔羊胴体品质没有影响,SO的添加可影响羔羊血清生化指标。
试验二研究了杭白菊茎叶对湖羊断奶羔羊生长性能、血清生化指标和胴体品质的影响。试验选取健康断奶湖羊羔羊36只(16+2.6kg),依据随机区组设计和同栏大小相近原则,根据体重分成4个处理,每个处理3个重复(栏),每个栏3只羔羊。试验采用精粗比为30：70的全混合颗粒饲粮,四组粗料中稻草和杭白菊茎叶(CSL)的比例分别为70:0 (CSLO),45:25 (CSL25),25:45 (CSL45)和0：70(CSL70)。试验结果,随着CSL饲喂比例的增加,羔羊的平均日增重增加,血清尿素氮(BUN)含量提高,CSL0.组和CSL70组间差异达显著水平(P<0.05)。CSL对羔羊血清过氧化氢酶(CAT)、谷胱甘肽过氧化物酶(GSH-Px)活力、总抗氧化能力(T-AOC)和血清丙二醛(MDA)含量均有不同程度的影响。随着CSL饲喂比例增加,羔羊血清中CAT和T-AOC增加,CSL45组和CSL70组与CSLO组相比,差异显著(P<0.05)；CSL45组GSH-Px活力显著高于其余各组(P<0.05)；MDA随CSL饲喂比例的增加而减少,CSLO和CSL70组间差异显著(P<0.05)。CSL的饲喂对羔羊胴体品质不存在显著影响,但是屠宰率CSL70组显著高于CSLO组(P<0.05)。上述结果表明,CSL用作粗料饲喂断奶湖羊可以提高抗氧化能力,促进生长。
第二部分植物源性物质对生长湖羊瘤胃发酵的影响(试验三和试验四)
试验三研究茶皂素和豆油对羔羊瘤胃发酵参数以及瘤胃微生物区系的影响。待饲养试验结束后,动物屠宰后取瘤胃液样品,测定瘤胃发酵参数及瘤胃微生物区系。结果显示：与NTNS组相比,TS,SO和TS-SO组的瘤胃pH值均显著降低(P<0.05)。添加TS和SO显著提高总挥发性脂肪酸(VFA)含量(P<0.05),但是对单独的VFA不存在显著影响(P>0.05)。氨态氮的含量在SO和TS-SO组显著低于NTNS组(P<0.05),但是TS组变化不大。与NTNS组相比,TS,SO和TS-SO组的微生物蛋白的含量均显著提高。添加TS和SO对真菌数量没有显著影响(P>0.05),TS作为主效应对原虫数量影响不显著,但是通过二次分析发现,TS对原虫的作用取决于SO,单独添加效果是显著的(P=0.04),但是与SO同时添加效果则不显著(P=0.68)。添加SO显著降低甲烷菌的数量(P<0.05)。SO的添加显著降低黄色瘤胃球菌和产琥珀酸丝装杆菌的数量(P<0.05),但是TS对这两个纤维菌没有影响。从DGGE图谱来看,日粮中添加TS和SO对瘤胃细菌、原虫以及甲烷菌区系有一定的影响,但是各组间条带数、多样性指数不存在显著差异(P>0.05)。结论：添加茶皂素和豆油对羔羊瘤胃微生物数量和微生态区系均有一定影响。
试验四研究杭白菊茎叶对湖羊瘤胃发酵参数以及氢化相关微生物的影响。瘤胃液取自试验二。试验结果显示：CSL70组湖羊瘤胃氨态氮的含量显著高于其余各组(P<0.05),微生物蛋白的含量CSL45组和CSL70组显著高于CSLO组和CSL25组(P<0.05)。瘤胃液相中,CSL的饲喂降低瘤胃原虫的数量,且在CSLO组和CSL70组间差异达显著(P<0.05)；溶纤维丁酸弧菌的数量CSL25组显著高于CSLO组和CSL70组(P<0.05)；蛋白溶解梭菌的数量CSL45组显著高于CSLO组(P<0.05)。瘤胃固相中,瘤胃原虫的数量也随着CSL的饲喂而减少,CSLO组和CSL45组间差异显著(P<0.05)；溶纤维丁酸弧菌的数量CSLO组显著高于CSL70组(P<0.05),但是蛋白溶解梭菌没有显著的变化。结果表明,杭白菊茎叶可影响瘤胃氨态氮和微生物蛋白的含量,以及瘤胃氢化菌的生长,这些影响可能与其所含有的黄酮类和挥发油类物质相关。
第三部分植物源性物质对生长湖羊肌肉脂肪酸组成及相关酶基因表达的影响(试验五和试验六)
试验五研究茶皂素和豆油对羔羊肌肉脂肪酸组成以及相关酶基因表达的影响。肝脏和肌肉样品取自试验一。试验结果显示：与NTNS组相比,各处理组肌肉组织中饱和脂肪酸(SFA)含量均有所降低,其中TS组和TS-SO组的C14：0与C15：0达显著水平(P<0.05)。各处理组肌肉组织中不饱和脂肪酸(UFA)的含量均有所增加,t11C18:1显著高于NTNS组(P<0.05)；SO组的cis-9, trans-11共轭亚油酸(CLA)显著高于其余各组(P<0.05)；SO组的多不饱和脂肪酸(PUFA)高于NTNS组(P<0.05)。日粮中添加TS和SO对羔羊肝脏中脂蛋白酯酶(LPL)基因的mRNA丰度不存在显著影响(P>0.05),但与NTNS组相比,TS和SO组肝脏组织中硬脂酰辅酶A去饱和酶(SCD)基因的mRNA丰度降低(P<0.05)。与NTNS组相比,TS和SO组肌肉组织中LPL基因mRNA丰度显著上升(P<0.05),且显著高于TS-SO组。肌肉组织中SCD基因mRNA丰度在SO组的表达显著高于其余各组(P<0.05)。因此推测添加SO对肌肉组织cis-9, trans-11CLA的影响受到SCD基因的调控。
试验六研究杭白菊茎叶对羔羊皮下脂肪和肌肉脂肪酸组成以及乙酰辅酶A羧化酶(ACC)基因在肝脏、皮下脂肪和肌肉组织中表达的影响。肝脏、皮下脂肪和肌肉样品取自试验二。结果显示：饲喂CSL可以提高羔羊皮下脂肪C16：1的含量,其中与CSLO组相比,CSL25组达显著水平(P<0.05)。C18：2的含量在CSL70组含量最高,显著高于CSLO组和CSL45组。总饱和脂肪酸和总不饱和脂肪酸随着CSL饲喂比例的增加而降低和增加,其中在CSLO组和CSL70组间差异达显著水平(P<0.05)。而饲喂CSL对羔羊肌肉中总不饱和脂肪和总饱和脂肪酸均没有显著影响(P>0.05)。CSL对肝脏、皮下脂肪和肌肉组织中ACC基因的表达均不存在显著影响(P>0.05)。ACC基因在肝脏中的表达量较少,比在脂肪和肌肉组织中表达量小2个数量级。结果表明：杭白菊茎叶的饲喂可影响动物脂肪和肌肉组织中脂肪酸的组成,但对ACC基因的表达没有影响。
综上所述,茶皂素和豆油的添加可以降低湖羊羔羊甲烷排放,从而减少饲料能的损失。日粮中添加茶皂素可能通过降低原虫数量从而降低羔羊肌肉组织中CLA的含量。添加豆油可降低肌肉组织中饱和脂肪酸,提高不饱和脂肪酸和CLA的含量,豆油对肌肉组织中cis-9, trans-11 CLA的合成可能是通过SCD基因调控实现。杭白菊茎叶饲喂羔羊可提高抗氧化能力,促进生长,且可通过杭白菊茎叶中的黄酮成分作用于瘤胃氢化菌而影响脂肪和肌肉组中的脂肪酸的组成,降低羔羊皮下脂肪组织中总饱和脂肪酸,增加不饱和脂肪酸含量,及影响肌肉组织中C18：0和C18：2的含量。
The components of plant orgin have many active properties, such as antibacterial and antioxidant. Thus, they couled be used in animal feed to replace the antibiotic. This study contained three parts including six experiments and it was conducted to investigate the effect of tea saponins (TS), soybean oil (SO) and chrysanthemun stem-leaf (CSL) on carcass quality, rumen fermentation and fatty acid profiles of muscle in growing lambs and approach to the mechanism involved.
Part I:Effect of components of plant origin on carcass quality of growing lambs. (Expt.1 and 2)
Experiment 1 was carried out to investigate the effects of TS, SO, and TS-SO on serum parameters and carcass quality of growing lambs. Thirty-two Huzhou lambs weaned at the age of 50 days, with an initial body weight of 14.2±1.38 kg, were assigned to four dietary treatments in a 2×2 factorial arrangement with TS (0 or 3g/d) and SO (0 or 3% of DM). The diet without additives was considered as NTNS (no TS or SO). Feed intake was controlled to be similar among the four groups to minimize the meat differences resultant from growth rate. After the feeding trial, four lambs from each treatment were moved to simple open-circuit respiratory chambers (two animals per chamber) to measure methane production. At the end of methane measurement, blood samples of all animals were taken via jugular vein puncture and serums were separated from the blood samples for analysis. Then animals were slaughtered and carcasses were weighed and dissected. No differences were observed in animal growth performances, carcass qualities among four treatments (P> 0.05). Addition of tea saponins had no effect on the serum parameters. Compared with NTNS, the contents of serum total protein, high density lipoprotein-cholesterol, cholesterol and non-esterified fatty acid were higher but the concentration of glucose was lower in SO and TS-SO groups than in NTNS group (P< 0.05). The content of low density lipoprotein-cholesterol was higher in SO group than in TS group (P< 0.05). These results indicated that addition of TS and SO can suppress the methane emission to improve the efficiency of feedstuff utilization, but have little effect on the carcass qualities of lambs.
Experiment 2 was conducted to determine the effect of feeding CSL on growth performance, serum parameters and carcass quality of growing lambs. Thirty-six Huzhou growing lambs were assigned to four groups of nine lambs each, and allotted to three units of three lambs at similar weight. The ratio of roughage to concentrate in all the diets was 70:30. Proportion of rice straw to CSL in roughage was 70:0 (CSLO),45:25 (CSL25),25:45 (CSL45), and 0:70 (CSL70) in diets, respectively. The trial lasted for 72 days with the first 12 days for adaptation. Feed ingested and refused were recorded every week, and body weight gain was measured every two weeks. At the end of the trial, serum biochemical and antioxidant parameters and carcass quality were determined. With increasing levels of CSL, average daily gain and serum urea nitrogen increased, with a significant difference between diets CSLO and CSL70 (P< 0.05). Serum catalase and total antioxidant capacity were significantly higher (P <0.05) in lambs on diets CSL45 and CSL70 than those on diet CSLO. The level of serum glutathione peroxidase was highest in animals on diet CSL45 (P< 0.05). Compared to that in diet CSLO, serum malondialdehyde was decreased in diet CSL70 (P< 0.05). Chrysanthemum stem-leaf had no effect on the carcass quality, but the dressing percent was higher in group CSL70 than in group CSLO (P< 0.05). From the present study, it is inferred that inclusion of CSL in the diets could increase antioxidant ability in growing lambs and improve growth performance.
Part II:Effect of components of plant origin on rumen fermentation and microbial populations in the rumen of lambs. (Expt.3 amd 4)
In Experiment 3, rumen fluids were sampled from the sheep used in Expt. 1 to determine the effects of addition of TS and SO on rumen fermentation and microbial populations in the rumen of growing lambs. The microbial communities were analyzed by real-time PCR and denaturing gradient gel electrophoresis technique. Populations of rumen methanogens, protozoa, fungi, Ruminococcus flavefaciens, and Fibrobacter succinogenes were expressed as a proportion of total rumen bacterial 16S rDNA. Ruminal pH was decreased (P< 0.05) for lambs fed diets with TS, SO, and TS-SO, vs. the NTNS, and ammonia N concentration was reduced by SO (P< 0.05). Concentrations of total volatile fatty acids were increased in all groups (P< 0.05), with no significant differences in proportions of individual acids among diets. Microbial protein was increased (P< 0.05) with TS, SO, and TS-SO. Addition of TS, SO, and TS-SO had little effect on fungal population (P> 0.05), but protozoa populations relative to total bacterial 16S rDNA were decreased (P< 0.05) for lambs fed diets with TS, SO, and TS-SO, with the lowest value in lambs fed the diet containing SO only. Population of methanogens was inhibited by SO (P< 0.05), but not by TS. Addition of SO and TS-SO had an inhibitory effect on the population of fibrolytic microbes including R. flavefaciens and F. succinogenes. Addition of TS and SO has an effect on the communities of bacterial, protozoa and methanogens. However, the diversities of bacterial, protozoa and methanogens were not affected by TS and SO. From the present study, it is inferred that the rumen microbial communities could be affected by addition of tea saponin and soybean oil.
Experiment 4 was conducted to determine the effect of feeding CSL on rumen fermentation and microbial populations in the rumen of growing lambs. The rumen samples both liquid and solid were taken from the animals in experiment 2. Ammonia N concentration was lowest in CSL70 (P< 0.05) and microbial protein was higher in CSL45 and CSL70 than in CSL0 and CSL25 (P < 0.05). In the liquid of rumen fluid, CSL decreased the protozoa populations; the population of B.fibrisolvens was higher in CSL25 than in'CSLO and CSL70 (P < 0.05); the C.proteoclasticum population was greater in diet CSL45 than in diet CSL0 (P< 0.05). In the solid of rumen fluid, the population of protozoa was decreased in diet CSL45 as compared to in the diet CSL0 (P< 0.05); the B.fibrisolvens population was larger in diet CSL0 than in diet CSL70 (P< 0.05); But the population of C.proteoclasticum was uninfluenced by CSL. It is inferred that the rumen fermentation and microbial populations could be affected by the flavone and naphtha components of CSL.
Part III:Effect of components of plant origin on fatty acid composition in longissimus dorsi muscle and expression of related enzyme gene. (Expt.5 and 6)
In Experiment 5, the longissimus dorsi (LD) muscle and liver tissuses were taken from the animals in Experiment 1 to determine the effects of addition of TS and SO on fatty acid profile in LD muscle, gene expression of lipoprotein lipase (LPL) and stearoyl-CoA desaturase (SCD) both in liver and muscle of growing lambs. Vaccenic acid percentage was enhanced (P< 0.05) in muscle of lambs fed TS and SO. The proportion of cis-9, trans-11 conjugated linoleic acid (CLA) was increased (P< 0.05) by SO, but decreased (P< 0.05) by addition of TS in LD muscle. The percentage of total saturated fatty acids (SFA) in muscle was decreased (P< 0.05) by addition of TS and SO, while addition of SO increased (P< 0.05) the percentage of total polyunsaturated fatty acids (PUFA). The ratio of cis-9, trans-11 CLA to vaccenic acid was decreased (P< 0.05) by TS, but increased (P< 0.05) by SO. Addition of TS and SO had no effect on the LPL gene expression in liver (P> 0.05). Compared with NTNS, the SCD mRNA expression in liver was increased in group TS and SO. The LPL mRNA expression in muscle was higher in group TS and SO than in NTNS. Addition of SO could increase the SCD mRNA expression (P< 0.05). These results indicated that inclusion of TS and SO in the diet of growing lambs affect the fatty acid profiles of LD muscle and that the proportion of cis-9, trans-11 CLA in the muscle influenced by TS and/or SO, may be regulated by SCD gene.
Experiment 6:This study was conducted to determine the effect of feeding CSL on fatty acid profile in LD muscle and subcutaneous fat, Acetyl CoA carboxylase (ACC) mRNA expression in liver, subcutaneous fat, and LD muscle. The liver, subcutaneous fat and LD muscle were taken from the animals in Experiment 2. In subcutaneous fat, the content of C 16:1 was increased by CSL; there was a highest proportion of C18:2 in diet CSL70 (P< 0.05); with increasing levels of CSL, the total SFA were decreased but the total undaturated fatty acids (UFA) were increased (P< 0.05). However, CSL did not affect the total SFA and total UFA in LD muscle. Inclusion of CSL had no effect on ACC mRNA expression in liver, subcutaneous fat, and LD muscle (P> 0.05). The ACC gene expression in liver was lower than in subcutaneous fat, and LD muscle. These results indicated that the fatty acid composition of subcutaneous fat and LD muscle could be affected by CSL, which may not be related to the ACC gene expression.
In summary, addition of TS and SO could reduce methane production to enhance the efficiency of feedstuff utilization. The intramuscular fat of lambs fed a diet containing TS had lower proportion of CLA. Dietary addition of linoleic acid-rich SO was effective in decreasing SFA proportion and increasing the proportion of PUFA and cis-9 trans-11 CLA in the LD muscle, which may be mediated through the expression of SCD gene. Inclusion of CSL in the diets could increase antioxidant ability in growing lambs and improve growth performance. The CSL could reduce the proportion of total SFA and increase the percentage of total UFA in subcutaneous fat, which may be related to the biohydrogenation associated bacteria in rumen affected by flavone in CSL,
引文
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