| 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.
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