【作者】 刘立成；

【导师】 刘大森；

【作者基本信息】 东北农业大学 ， 动物营养与饲料科学， 2009， 博士

【摘要】 本研究旨在通过体外和体内试验评价植物油和瘤胃调控剂(莫能菌素)的复合使用对提高CLA及其前体物的累积效果。体外部分是利用气相色谱法测试植物油中脂肪酸的含量,通过体外氢化试验筛选出合成脂肪酸效果最好的植物油及适宜的莫能菌素添加范围;体内部分根据体外试验结果将豆油和莫能菌素应用到动物试验中,研究其对瘤胃发酵、脂肪酸合成、日粮消化和血液参数的影响。体内试验以豆油和莫能菌素为两个试验因子,选择4只体况良好、健康无疾病体重38±1kg安装有永久性瘤胃瘘管的绵羊为试验动物,采用4×4拉丁方试验设计,共分4期,每期12d,分别向动物基础日粮中添加0%(对照组,CK),4%豆油(豆油组,S组),4%豆油加25mg/kg的莫能菌素(复合组,S+M组),25mg/kg莫能菌素(莫能菌素组,M组)。试验一、采用毛细管气相色谱法测试常见植物油中脂肪酸的组成。测试仪器为岛津GC-2010气相色谱仪,SP-2560(100m×0.25mm×0.2μm)色谱柱,载气高纯氮气(99.999%),FID检测器240℃,进样口温度240℃,分流比50:1。柱温箱二阶段程序升温:初始温度165℃,维持30min,以1.5℃/min速率升至200℃保持20min,再以5℃/min升到到230℃,维持5min。压力:266.9kPa,柱流量1.04mL/min,线速度20cm/s。结果表明,毛细管气相色谱法能很好的分离植物油中各种脂肪酸,豆油、葵花油和玉米油中油酸和亚油酸百分含量比分别为28:55、27:61和22:54,但玉米油中含有4%的亚麻酸;花生油中油酸和亚油酸百分含量比为46:33,橄榄油属于高油酸植物油,其油酸含量为78%,亚油酸为5.7%;亚麻油属高亚麻酸类植物油,其中油酸:亚油酸:亚麻酸百分含量比为21:14:54。试验二、体外培养条件下植物油添加量的筛选。培养底物为0.5g微晶纤维素,培养液体积为60mL,培养瓶中植物油的添加水平分别为0、5、10、15和20mg,每个水平培养时间点分别为4、8、16、24、36和48h,每个时间点设4个重复。结果表明:随着植物油添加水平的逐渐增加,培养液中pH值呈上升趋势,NH3-N浓度、TVFA浓度和48h纤维素消失率逐渐降低。在本试验培养条件下,保证瘤胃正常发酵的植物油最适宜添加量为5mg,即脂肪添加量占底物的1%。试验三、不同植物油体外发酵及PUFA(多不饱和脂肪酸)合成规律的研究。根据试验二筛选出的植物油添加量,选择豆油、玉米油、葵花油和花生油做脂肪源,培养时间点分别为2、4、8、16和24h,测试各时间点PUFA氢化及t11-C18:1累积。随着培养时间的延长,培养液中C18:0含量逐渐增加,c9,c12-C18:2含量逐渐降低,t11-C18:1含量先升高而后降低再升高,在24h达到最大值;添加植物油显著提高了培养液中C18脂肪酸的含量(P<0.05);培养液中t11-C18:1含量比较,豆油组最高(P<0.05),其次是葵花油(P<0.05),花生油最低;玉米油组c9,c12-C18:2含量最高(P<0.05),其次是豆油组(P<0.05),花生油组最低。综合比较四种植物油PUFA氢化及t11-C18:1累积,豆油作为脂肪源效果最好。试验四、不同水平的莫能菌素对亚油酸体外发酵及PUFA合成规律的研究。以游离脂肪酸(亚油酸)为脂肪源,微晶纤维素为培养底物,莫能菌素的添加量分别为0、10、20、30和40mg/kg底物,培养时间点分别为2、4、8、16和24h,测试各时间点瘤胃发酵及PUFA氢化规律。结果表明:添加莫能菌素能降低培养液pH值,提高NH3-N和TVFA浓度,且莫能菌素添加量与纤维素消失率呈二次函数关系,关系式为y=-0.0261x2+1.0651x+7.6355,相关系数为R2=0.9918,通过导函数可以计算出当莫能菌素添加量超过40.8mg/kg时抑制纤维素的消化。随着培养时间的延长培养液中C18:0含量逐渐增加,且添加莫能菌素能显著降低培养液中C18:0含量(P<0.05);莫能菌素的添加量与t11-C18:1含量呈二次函数关系,关系式为y=0.0014X2-0.0266X+0.3747,相关系数为R2=0.9894,通过导函数可以计算出当莫能菌素添加量超过9.5mg/kg时,培养液中t11-C18:1随着莫能菌素添加量的增加呈逐渐升高的趋势。莫能菌素能显著降低培养液中c9-C18:1含量(P<0.05),但莫能菌素添加量与c9-C18:1合成无剂量效应。莫能菌素能抑制c9,c12-C18:2氢化,提高了培养液中c9,c12-C18:2含量,且高剂量莫能菌素抑制效果显著(P<0.05)。随着莫能菌素添加量的增加t11-C18:1和c9,c12-C18:2氢化率逐渐降低。本试验根据瘤胃发酵和PUFA合成确定莫能菌素添加范围为19~40.8mg/kg。试验五、豆油和莫能菌素对瘤胃脂肪酸合成规律的影响。添加豆油显著提高了瘤胃液中C16:0和C18:0含量(P<0.05),而添加莫能菌素显著降低了瘤胃液中C16:0和C18:0含量(P<0.05),与豆油组相比较,豆油和莫能菌素复合处理组降低了C16:0和C18:0含量(P<0.05)。日粮中添加豆油瘤胃液中t11-C18:1和c9-C18:1显著增加(P<0.05),添加莫能菌素组t11-C18:1含量显著增加(P<0.05),而c9-C18:1含量降低,与豆油组相比较,豆油和莫能菌素复合处理组t11-C18:1显著增加(P<0.05),说明莫能菌素对由豆油合成t11-C18:1具有促进作用。添加豆油和莫能菌素各组均显著提高了瘤胃液中c9,c12-C18:2和c9,t11-CLA(P<0.05),且随着饲喂时间的延长先升高后降低,c9,c12-C18:2和c9,t11-C18:2含量在饲喂后的4h达到最大值。添加莫能菌素的两组瘤胃液中t11-C18:1、c9,c12-C18:2和c9,t11-CLA氢化率降低,而添加豆油组提高了c9,c12-C18:2氢化率。本试验结果表明,添加豆油能提高瘤胃脂肪酸的含量,添加莫能菌素能降低瘤胃饱和脂肪酸的含量,提高PUFA尤其是t11-C18:1的含量,且豆油和莫能菌素复合使用效果更佳。试验六、添加豆油和莫能菌素提高了瘤胃液pH值和BCP浓度(P>0.05),降低了NH3-N浓度(P>0.05);豆油组显著降低瘤胃液中TVFA浓度(P<0.05),添加莫能菌素组有提高TVFA浓度的趋势(P>0.05),添加莫能菌素组显著降低瘤胃液中乙酸浓度(P<0.05),单独添加莫能菌素组显著提高瘤胃液中丙酸浓度(P<0.05)。添加豆油和莫能菌素都降低瘤胃中纤维素酶和脂肪酶活性。综合瘤胃发酵指标,豆油和莫能菌素复合处理不会降低瘤胃pH、NH3-N、BCP和VFA,但对微生物酶活性有降低趋势。试验七、日粮中添加豆油血液中的TG、CHOL、HDL和LDL相应的增加,添加莫能菌素显著提高血液中Leptin的水平(P<0.05);豆油和莫能菌素有提高血液中GLU和NEFA的趋势,但差异不显著(P>0.05),添加豆油显著提高血液中T-AOC和SOD含量(P<0.05),降低了MDA含量;豆油和莫能菌素复合处理能提高机体抗氧化水平。日粮中添加豆油和莫能菌素显著降低血液中16和18碳饱和脂肪酸含量(P<0.05),提高t11-C18:1含量(P<0.05);且豆油和莫能菌素复合处理组显著提高血液中t11-C18:1和c9,c12-C18:2含量(P<0.05)。试验八、添加豆油和莫能菌素对瘤胃液相流通规律和DM降解率没有影响(P>0.05),添加豆油显著降低瘤胃CP降解率(P<0.05),莫能菌素能显著降低日粮ADF降解率(P<0.05),添加豆油和莫能菌素均能显著提高NDF降解率。添加豆油和莫能菌素有降低日粮营养物质表观消化率的趋势,单独添加莫能菌素能显著提高日粮CP表观消化率(P<0.05)。本试验结果表明,莫能菌素和豆油复合处理不会降低日粮消化率和降解率,说明复合处理不会营养日粮的消化。更多还原

【Abstract】 The objective of this thesis was to evaluate the compound use of vegetable oil and rumen adjusting agent on improving CLA content in vitro and in vivo. The in vitro experiment was conducted to determine the fatty acid content in vegetable oils using gas chromatograpgy, and select the best vegetable oil for the synthesis of fatty acid and the suitable addition of monensin. The in vivo experiment was conducted to evaluate the effect of soybean oil and monensin which were applied as two testing factors on rumen fermentation, fatty acid composition, diet digestibility, and blood parameters. Four healthy sheep (BW=38±1kg) fitted with permanent ruminal cannulas were assigned to 4 dietary treatments in a 4×4 Latin square over four consecutive periods of 12d each. 4 sheep received either a control diet or one of 3 treatment diets. The control diet (CK) consisted of 60% forage and 40% concentrate on a air-dry matter basis. The treatments diets were formulated with supplemental 4% of soybean oil (S), 4% of soybean oil plus 25mg/kg of monensin (S+M), or 25mg/kg monensin (M).Experiment 1, The objective of this study was to determine the fatty acid composition in vegetable oils using capillary gas chromatograpgy. Vegetable oils were analyzed for FA on a Shimadzu GC-2010 gas chromatograph equipped with a flame-ionization detector and 100-m SP-2560 fused silica capillary column (100m×0.25mm×0.2μm). The injector and detector temperatures were set at 240℃, and the split ratio in the injector port was 50:1. Purified nitrogen was used as the carrier gas with a head pressure of 266.9 kPa, a flow rate of 1.04mL/min, and linear velocity of 20cm/s. The initial column temperature was set at 165℃and held for 30min, increased to 200℃at 1.5℃/min and held for 20min, further increased to 230℃at 5℃/min, and finally held at 230℃for 5min. The result showed that the fatty acid in vegetable oils could be separated by capillary gas chromatograpgy very well. The ratio of oleic acid to linoleic acid was 28:55, 27:61, or 22:54 in soybean oil, sunflower oil, or corn oil respectively. There was 4% of linolenic acid in corn oil. The ratio of oleic acid to linoleic acid was 46:33 in peanut oil. Olive oil was rich in oleic acid with the percentage of 78%, 5.75% of linoleic acid. Linseed oil was rich in linolenic acid, and the ratio of oleic acid to linoleic acid to linolenic acid was 21:14:54.Experiment 2, The objective of this study was to determine the addition of vegetable oil in vitro. Avicel was used as substrates, and vegetable oil was supplemented at the level of 0, 5, 10, 15 and 20mg respectively of 0.5g avicel. The incubation time was 4, 8, 16, 24, 36 and 48h for each level, and set 4 repetition for each incubation at each time. Results showed that the pH showed an increasing tendency with the increasing addition of vegetable oil,, but the NH3-N concentrations, the TVFA concentrations, and the cellulose disappearance rate at 48h decreased gradually. Under this culture condition, the best addition of vegetable oil was 5mg to ensure the normal rumen fermentation, that is the fat supplementation accounts for equal to 1% of the substrate.Experiment 3, The objective of this study was to determine the ruminal fermentation of different vegetable oils and the synthesis regularity of PUFA (polyunsaturated fatty acids). The addition of vegetable oils was determined by experiment 2, and soybean oil, corn oil, sunflower oil, and peanut oil were used as fat sources. The incubation time was 2, 4, 8, 16, and 24h respectively, and culture fluid was used to determine the concentrations of PUFA and t11-C18:1. With the increasing of incubation time, the C18:0 content increased , the c9,c12-C18:2 content decreased gradually in culture fluid, and the content of t11-C18:1increased at first and decreased subsequently and then increased again with the maximum value at 24h. Contents of C18 fatty acids were increased (P<0.05) significantly in culture fluid with vegetable oils. The soybean oil treatment had the maximum content of t11-C18:1 (P<0.05), then sunflower oil treatment (P<0.05), and peanut oil treatment was lowest. The maximum content of c9,c12-C18:2 appeared in corn oil treatment (P<0.05), then soybean oil treatment (P<0.05), the lowest content appeared in peanut oil treatment. Comprehensive comparison the 4 vegetable oils, soybean oil as fat source had the best effect on the biohydrogenation of PUFA and the accumulation of t11-C18:1.Experiment 4, The objective of this study was to determine the effect of monensin at different levels on ruminal fermentation of linoleic acid (LA)and synthesis regularity of PUFA in vitro. LA was used as fat source and avicel was used as substrates. Monensin was supplemented at the level of 0, 10, 20, 30, and 40mg/kg substrates. After 2h, 4h, 8h, 16h, and 24h of incubation respectively, ruminal fermentation parameters and the biohydrogenation of PUFA in the culture fluid were determined. Results showed that monensin could decrease rumen pH and increase concentrations of NH3-N and TVFA in culture fluid, and there was a quadratic function relationship between the addition of monensin and the disappearance rate of cellulose. The relationship formula was y=-0.0261x2+1.0651x+7.6355, with R2=0.9918. It could be calculated by the derivative function that the disappearance rate of cellulose was inhibited while the addition of monensin was beyond 40.8mg/kg. The content of C18:0 in culture fluid increased with the increasing of incubation time, and decrease (P<0.05) with the supplementation of monensin. The quadratic function relationship between the addition of monensin and the content of t11-C18:1 was y=0.0014X2-0.0266X+0.3747, with R2=0.9894. It could be calculated by the derivative function that the content of t11-C18:1 in culture fluid increased with the increasing addition of monensin while the addition of monensin was beyond 9.5mg/kg. Monensin could decrease (P<0.05) the content of c9-C18:1, but there was no dose-effect between the addition of monensin and the synthesis of c9-C18:1. Monensin could inhibit the biohydrogenation of c9,c12-C18:2 and improve the content of c9,c12-C18:2 in culture fluid, furthermore the high dosage of monensin had the obvious inhibitory effect (P<0.05). The biohydrogenation rate of t11-C18:1 and c9,c12-C18:2 decreased with the increasing addition of monensin. It was concluded that the addition range of monensin was 9.5~40.8mg/kg in this experiment by ruminal fermentation and the synthesis of PUFA.Experimemt 5, The objective of this study was to determine the effect of monensin and soybean oil on fatty acid composition. Soybean oil increased (P<0.05) the contents of C16:0 and C18:0 in rumen fluid, but monensin decreased (P<0.05) the contents of C16:0 and C18:0 in rumen fluid. S+M treatment decreased (P<0.05) the contents of C16:0 and C18:0 in rumen fluid compared with S treatmeat. Soybean oil increased (P<0.05) the contents of t11-C18:1 and c9-C18:1 in rumen fluid and monensin increased (P<0.05) the contents of t11-C18:1, but decreased (P<0.05) the contents of c9-C18:1 in rumen fluid. S+M treatment increased (P<0.05) the content of t11-C18:1 in rumen fluid compared with S treatmeat. It was concluded that monensin could promote the synthesis of t11-C18:1 when diet was supplementel with soybean oil. Dietary supplementation with soybean oil and monensin increased (P<0.05) the the contents of c9,c12-C18:2 and c9,t11-CLA in rumen fluid, and the contents increased at first and decreased subsequently with feeding time and the maximum appeared at 4h after feeding. The biohydrogenation rate of t11-C18:1, c9,c12-C18:2,and c9,t11-CLA in the rumen fluid of dietary supplemented with monensin, but dietary supplemented with soybean oil decreased the biohydrogenation rate of c9,c12-C18:2. Results showed that dietary supplemented with soybean oil could increase the content of rumen fatty acid, and with monensin could decrease the content of saturated fatty acids and increase the content of PUFA especially t11-C18:1, furthermore, the compound use of soybean oil and monensin had a better effect.Ecperiment 6, Dietary supplementation with soybean oil and monensin increased (P>0.05) ruminal pH and the concentration of bacterial protein and decreased (P>0.05) the concentration of NH3-N. The concentration of TVFA in rumen fluid was decreased (P<0.05) in S treatment. Diet supplemented with monensin had a trend of increasing (P>0.05) the TVFA concentration, and decreased (P<0.05) the acetate concentration. The M treatment increased (P<0.05) the concentration of propionate. Dietary supplementation with soybean oil or monensin could decrease the activities of celulase and lipase.Experiment 7, Dietary supplementation with soybean oil could increase the concentration of TG, CHOL, HDL, and LDL in blood, and with monensin could increase (P<0.05) the level of leptin. Dietary supplementation with soybean oil and monensin had a trend of increasing the concentration of GLU and NEFA. Dietary supplementation with soybean oil increased (P<0.05) the concentration of T-AOC and SOD and decreased the concentration of MDA. The compound use of soybean oil and monensin could increase the antioxidation ability. Dietary supplementation with soybean oil and monensin decrease (P<0.05) the contents of C16 and C18 saturated fatty acid, and increased (P<0.05) the content of t11-C18:1 in blood. The compound use of soybean oil and monensin increaseed (P<0.05) the contents of t11-C18:1 and c9,c12-C18:2 in blood.Experiment 8, Dietary supplementation with soybean oil and monensin did not effect the passage rate and the degradation rate of DM and increased the degradability of neutral detergent fiber, but with soybean oil decreased (P<0.05) the effective degradability of crude protein, and with monensin decreased (P<0.05) the degradability of acid detergent fiber. Dietary supplementation with soybean oil and monensin had a trend of decreasing the whole tract apparent digestibility of dietary nutrients, but with monensin alone could increase (P<0.05) the apparent digestibility of crude protein.更多还原