【作者】 张文娟；

【导师】 欧阳五庆；

【作者基本信息】 西北农林科技大学 ， 细胞生物学， 2010， 博士

【摘要】 目的:通过纳米技术将脂溶性维生素A、D、E、K和水溶性维生素B1、B2、B6、B12、叶酸、生物素、泛酸钠、烟酰胺复合制成纳米乳,并对其功效进行评价,以期开发一种安全、高效、稳定、质量可控的禽用复合维生素纳米乳。方法:(1)通过伪三元相图,筛选并优化处方,参照家禽营养参数及饲养标准,确定各种维生素的比例,制备复合维生素纳米乳;用染色法鉴定纳米乳的类型,用透射电子显微镜和激光粒度分析仪,考察其形态和粒径分布;用高效液相色谱法检测其中8种维生素的含量;用光加速试验、温度加速试验以及长期试验,考察其稳定性;用雏鸡的急性毒性试验和亚急性毒性试验考察其安全性。(2)选用爱拔益加肉仔鸡、海兰褐商品蛋鸡、罗曼父母代种鸡作为试验动物,试验分为5组,分别为试验I、II、III、IV、V组,I组为复合维生素纳米乳高剂量组(1 000倍稀释),II组为复合维生素纳米乳中剂量组(2 000倍稀释液),III组为复合维生素纳米乳低剂量组(5 000倍稀释液),IV组为阳性对照组(普通复合维生素组),V组为阴性对照组,每种动物给予相同的基础日粮,分别研究复合维生素纳米乳及普通复合维生素对其生产性能和免疫功能的影响。(3)采用胰蛋白酶消化法分离鸡胚肝细胞,用台盼蓝拒染法检测肝细胞即时存活率;用倒置显微镜观察肝细胞的形态;通过MTT法检测复合维生素纳米乳及普通复合维生素对肝细胞增殖率的影响,并绘制生长曲线;用试剂盒检测肝细胞培养上清中白蛋白含量和丙二醛含量的变化,研究复合维生素纳米乳及普通复合维生素对肝细胞增殖和代谢的影响。结果:(1)制备的复合维生素纳米乳为水包油(O/W)型橙红色透明液体,流动性好,乳滴呈球形,分布均匀,平均粒径为29.8 nm;高效液相色谱检测复合维生素纳米乳中8种维生素的含量均符合配方设计要求;在光照(4 500±500)Lx条件下,放置0、5、10 d后,纳米乳颜色加深,其中维生素B1、B2和B6含量有下降趋势,而粒径变化不明显;在4℃、25℃、40℃、60℃条件下,放置30 d,纳米乳粒径有增大趋势,维生素含量有下降趋势;在常温下放置12个月,纳米乳粒径有增大趋势,维生素含量有下降趋势;安全性试验显示复合维生素纳米乳对雏鸡无急性毒性和亚急性毒性。(2)肉鸡试验结果显示:与V组相比,42日龄时I组肉鸡的死淘率降低87.50 %,料重比降低12.11 %,出栏重提高3.80 %,均差异显著(P<0.05);42日龄时,与V组相比,I组肉鸡血清中白蛋白含量增加45.66 %,碱性磷酸酶活性升高10.70 %,差异均显著(P<0.05);与V组相比,I、II、III组的半净膛率分别提高12.40 %、11.38 %、2.95 %,且差异显著(P<0.05);I组的全净膛率、胸肌率、腿肌率比IV组分别提高16.28 %、6.52%、5.95 %,且差异显著(P<0.05);ANAE+淋巴细胞比例I组与V组相比21日龄时提高5.30 %,42日龄时提高5.20 %,且差异显著(P<0.05),而与IV组相比差异不显著(P>0.05)。蛋鸡试验结果显示:与V相比,I、II、III组提高商品蛋鸡的产蛋量和产蛋率,降低料蛋比,其中I组产蛋量提高5.89 %,产蛋率提高5.33 %,料蛋比降低4.05 %,均差异显著(P<0.05),试验I、II的死淘鸡只数比IV、V组显著降低(P<0.05);试验I组的蛋黄颜色、蛋黄比例、蛋壳强度与IV、V组相比显著提高(P<0.05);I组和II组的门冬氨酸转移酶活性比V组分别提高57.52 %和45.70 %,碱性磷酸酶活性比V组分别提高40.95 %和36.09 %,均差异显著(P<0.05);与V组相比,I组和II组的总胆固醇含量分别提高87.62 %和75.25 %,且差异显著(P<0.05);试验I、II组的免疫器官指数与V组相比差异显著(P<0.05)。种鸡试验结果显示:与V组相比,I、II、III组均可提高蛋种鸡的日只产蛋量和产蛋率,其中日只产蛋量分别提高10.96 %、7.07 %、5.09 %,产蛋率分别提高2.87 %、2.66 %、2.47 %,均差异显著(P<0.05);与IV组和V组相比,I组可使种蛋合格率提高4.35 %、5.93 %,受精率提高3.28 %、4.95 %,孵化率提高5.85 %、8.82 %,且差异显著(P<0.05);48周时,与V组相比,I、II组均可提高种公鸡的精液品质,精液量分别提高27.27 %和15.15 %,精子密度分别提高7.09 %和7.37 %,精子活力分别提高8.11 %和6.76 %,均差异显著(P<0.05);I、II、III组种公鸡血清中T浓度分别比V组提高2.79 %、2.17 %和1.55 %,均差异显著(P<0.05)。(3)分离的肝细胞即时存活率为95%,倒置显微镜下肝细胞呈圆形,立体感强,I～V组肝细胞的增殖率分别为29.84 %、25.72 %、19.14 %、10.44 %、14.07 %,其中I、II组与V组相比差异显著(P<0.05);各组肝细胞培养上清中白蛋白的分泌量第3天达到峰值,分别为3.46 g/L、3.11 g/L、3.36 g/L、2.71 g/L、2.65 g/L、2.12 g/L,其中I、II、III组与VI组相比差异显著(P<0.05),MDA的含量各处理组均低于空白对照组,且具有剂量依赖关系。结论:(1)制备的复合维生素纳米乳为O/W型橙红色透明液体,流动性好,乳滴呈球形,分布均匀,平均粒径为29.8 nm;(2)建立的同时检测多种维生素的HPLC方法专属性好,回收率、重复性和精密度均能满足要求,可分别用于该复合维生素纳米乳的质量控制;(3)复合维生素纳米乳在常温、避光条件下稳定性好,有效期为一年,其对雏鸡无急性毒性和亚急性毒性,在治疗剂量范围内安全性高,符合生产实践的要求;(4)复合维生素纳米乳与普通复合维生素相比,能够提高肉鸡、蛋鸡、种鸡的生产性能和免疫功能;(5)复合维生素纳米乳与普通复合维生素相比,能够促进体外培养鸡胚肝细胞增殖,提高白蛋白的分泌,降低培养上清液中MDA的含量。因此,复合维生素纳米乳具有安全、高效、稳定、质量可控的优点,是一种提高家禽生产性能和免疫力的新型复合维生素制剂。更多还原

【Abstract】 Objective: In order to develop one of security, effectiveness, stability, and quality controllable composite vitamin nanoemulsion for poultry, we compounded fat-soluble vitamins A, D, E, K and water-soluble vitamins B1, B2, B6, B12, folic acid, biotin, sodium pantothenate, nicotinamide and made it into nanoemulsion through nanotechnology and its effectiveness was evaluated.Methods: (1) We prapared composite vitamin nanoemulsion by optimizing prescription with drawing pseudo-ternary phase and confirming ratio of every vitamin according to animal nutrition parameter and feeding standards. We identified the nanoemulsion type by staining and studied the morphology and particle size distribution by transmission electron microscope and laser particle sizer. We examined content of vitamin A, D3, E, B1, B2, B6, nicotinamide and sodium pantothenate by High Performance Liquid Chromatography. We studied stability by accelerated test and long run test and security by acute toxicity test and sub-acute toxicity test in chicken. (2) We selected Arbor Acres broilers, Highland Brown commercial laying hens and Roman parents breeder as a test animal which were divided into five groups (group I, II, III, IV, and V), respectively. Group I, II, and III was high (1 000 times dilution), middle (2 000 times dilution), and low dose (5 000 times dilution) group of composite vitamin nanoemulsion, respectively. Group IV was positive control group (common composite vitamin). Group V was negative control group. Every kind of test animal was given the same basic diet. We investigated effects of composite vitamin nanoemulsion on productivity and immune function in broilers, laying hens and breeder. (3) We seperated the chick embryo hepatocytes by trypsin digestion and detected the immediate survival rate by trypan blue excluaion staining. The morphology was observated by inverted microscope. The effect of composite vitamin nanoemulsion and common composite vitamin on multiplication rate was detected by MTT. The albumin concents and MDA concents in mediums were detected by detection kit. We research the effect of composite vitamin and common composite vitamin on multiplication and metabolize of hepatocytes.Results: (1) The composite vitamin nanoemulsion was salmon pink transparent liquid with type of O/W and emulsion droplets were roundness and uniformly distributed with a mean diameter of 29.8 nm. The content of three kinds of fat-soluble VA, VD3, VE and five kinds of water-soluble VB1, VB2, VB6, nicotinamide and sodium pantothenate examined by High Performance Liquid Chromatography acetate were in accordance with requirement of prescription. In the light of (4 500±500) Lx conditions for 0, 5 and 10 d, the color of the composite vitamin were darken and there was a downward trend in the content of VB1, VB2, and VB6, while the nanoemulsion particle size did not change significantly. After 30 d storage under temperature of 4℃, 25℃, 40℃, and 60℃and 12 months storage under normal temperature, the particle size of nanoemulsion were increased slightly and the vitamin conent were depressed slightly. The results of acute toxicity test and sub-acute toxicity test show that the composite vitamin nanoemulsion was scurity for chicken. (2) The results in broiler experiment showed that: Compared with group V, in group I the death rate of broiler in 42 days old decreased 87.50 %, F/G decreased 12.11 % and the slaughter weight increased 3.80 %, there were significant difference (P<0.05); ALB content in serum of broiler of 42 days old increased 45.66 %, ALP activity increased 10.70 %, there were significant difference (P<0.05). Compared with group V, semi-eviscerated in group I, II, and III increased 12.4 %, 11.38 %, and 2.95 % and there were significant differcence (P<0.05). Compared with group IV, full-eviscerated in group I incresse 16.28 %, pectoral rate increased 6.52 % and thigh rate increased 5.95 %, and there were significant differcence (P<0.05). Compared with group V, ANAE+ lynphocyte rate increased 5.30 % in 21 days old and 5.20 % in 42 days old, respectively, and there were significant difference (P<0.05), but compared with group IV, there were no significant differcence (P>0.05). The results in laying hen experiment showed that: Compared with group V, daily egg production and laying rate of commercial laying hens in group I, II, and III all increased and feed egg ratio decreased in which dialy egg production in group I increased 5.89 %, laying increased 5.33 % and feed egg ratio decreased 4.05 %, which can been seen significant differcence (P<0.05). There were significant difference in death number of hens between group I, II and IV group (P<0.05). There were significant difference in yolk color, strength of eggshell and rate of yolk between group I and group IV and V (P<0.05). AST activity in group I and II increased 57.2 % and 45.70 % than group V and ALP increased 40.95 % and 36.09 %, and there were siginificant difference (P<0.05). Compared with group V, CHOL content in group I and II increased 87.62 % and 75.25 %, respectively, and there were siginificant difference (P<0.05). There were significant difference in the immune organ index between group I, II and group V (P<0.05). The results in breeder experiment showed that: Compared with group V, daily egg production in breeder of laying hens in group I, II, and III increased 10.96 %, 7.07 %, 5.09 % and laying rate increased 2.87 %, 2.66 %, 2.47 %, and there were significant difference (P<0.05). Compared with group IV and V, passing rate of egg in I group increased 4.35 % and 5.93 %, fertilization rate increased 3.28% and 4.95% and hatching rate increased 5.85 % and 8.82 %, and there were significant difference (P<0.05). Compared with group V, semen quality of 48-week rooster in I and II group increased significantly in which sperm volume increased 27.27 % and 15.15 %, sperm count 7.09 % and 3.37 %, sperm motility 8.11 % and 6.76 %, and there were significant difference (P<0.05). Compared with group V, T content in serum of roosters in group I, II, and III increased 2.79 %, 2.17 % and 1.55 %, and there were significant difference (P<0.05). (3) The results showed that the immediate survival rate of the chick embryo hepatocytes were 95%. The chick embryo hepatocytes was round and stereoscopic observed under the inverted microscope. The multiplication rates of different concentration of composite vitamin nanoemulsion were 29.84 %, 25.72 %, 19.14 %, 10.54 %, and 14.07 % respectively, and there were significant difference between group I, II and group V. The albumin contents in mediums of different treatment had reached peak on the third day and the contents was 3.46 g/L, 3.11 g/L, 3.36 g/L, 2.71 g/L, 2.65 g/L, 2.12 g/L respectively, and significant difference (P<0.05) were observed between group I, II, and III and group VI. MDA concents in mediums of different composite vitamin nanoemulsion were lower than the control group, and there were dose dependence.Conclusion: (1) The composite vitamin nanoemulsion was salmon pink transparent liquid with type of O/W and emulsion droplets were roundness and uniformly distributed with a mean diameter of 29.8 nm. (2) Methods establised to examined content of three kinds of fat-soluble vitamin and five kinds of water-soluble vitamin were specific and recovery, repeatability and precision can meet the requirements for quality control of the composite vitamin nanoemulsion, respectively. (3) The quality of composite vitamin nanoemulsion was very stability under normal temperature and dark condition with one year validity and the results of safety testing showed that there were no acute toxicity and sub-acute toxicity to chicken. The composite vitamin nanoemulsion was very security in the therapeutic dose range and the security was complied with the requirements of production practice. (4) Compared with common composite vitamin, the copmposite vitamin nanoemulsion can not only improve the production performance of broiler, laying hens, breeder, but also immune function. (5) Compared with common composite vitamin, the composite vitamin nanoemulsion can promote proliferation of chicken embryo hepatocytes, increased albumin secretion and decreased MDA content in culture supernatant. Therefore, the composite vitamin nanoemulsion for poultry have excellence of security, effectiveness, stability, and quality controllable which can be used to increase poultry production performance and immunity as a new composite vitamin preparations.更多还原