Preparation Of Tributyrin Nano-emulsion And Its Regulation Of Intestinal Health In Broilers

Tributyrin as a butyric acid derivative has a promising application in poultry production.Tributyrin were added directly to the basal diet to improve the growth and development of broilers by promoting intestinal development and regulating gut health.Due to the poor water solubility and dispersibility,tributyrin is not conducive to digestion and absorption in broilers by direct feeding,while the intestinal function of broilers is not well developed during the hatching and pre-shelling period,and their digestion and absorption of nutrients is insufficient.To improve the disadvantages of insufficient energy supply and weak intestinal digestion and absorption in broilers during the incubation period.This study was conducted to improve the water solubility,dispersibility and stability of tributyrin by optimizing the preparation of tributyrin nano-emulsion（TBNE）;analyzing the bioavailability of TBNE in vitro;further investigate the effects of TBNE on early growth performance,intestinal development,intestinal microenvironment and intestinal regulation of broilers in ovo feeding;while investigate the effect of resistance and repair ability of TBNE in broilers to lipopolysaccharide（LPS）stress were investigated in ovo,and the following results were obtained:The conditions for the preparation of TBNE were optimized by response surface methodology using sorbitol,tributyrin and modified phospholipids as independent variables and the Z-average particle size of the emulsion as response values.The optimal preparation process of TBNE was obtained as sorbitol:tributyrin:modified phospholipid（mass ratio）of9:5:1.The Z-average particle size,Zeta potential,conductivity,emulsion stability index（ESI）and emulsification activity index（EAI）of TBNE prepared under this condition were 250.02±13.73 nm,-40.23±1.39 m V,31.80±1.71μS/cm,848.00±69.03 min and 1.14±0.03 m²/g,respectively.The results were not significantly different from the predicted values（P>0.05）.Therefore,the particle size of TBNE prepared by this process was below 300 nm,which improved the stability of TBNE and the water solubility of tributyrin.In vitro results showed that the particle size of TBNE significantly increased during the gastric digestion（P<0.05）but the emulsion morphology was not destroyed and the ESI and EAI remained stable（P>0.05）.The conductivity and EAI of TBNE decreased with digestion time and the Zeta potential increased gradually（P<0.05）after entering the intestinal digestion stage,while TBNE was digested and broken down and had a higher content of free fatty acids compared with tributyrin（P<0.05）.This result indicated that TBNE remained stable in the stomach against acidic conditions and pepsin action.And TBNE was more easily dispersible and digestible when entering the intestine,which improving the bioavailability of tributyrin.A total of 480 Arbor Acres（AA）white feather broiler fertilized eggs were randomly divided into five treatment groups:blank,control（0 mg/egg）,2 mg/egg,4 mg/egg and 8mg/egg treatment.TBNE did not significantly effect on hatchability（P>0.05）;it significantly increased the body weight of broilers at 3,7 and 14 d（P<0.05）;it significantly increased the average daily feed intake（ADFI）and average daily weight gain（ADG）of broilers from 1 to14 d（P<0.05）;it significantly improve in broiler intestinal organ development,villi length（VH）,crypt depth（CD）and villi height/crypt depth（VH/CD）in each intestinal segment of the small intestine at 3,7 and 14 d（P<0.05）.In addition,TBNE significantly reduced the ileal p H of 3,7 and 14 d broilers（P<0.05）,significantly increased Lactobacillus live bacteria count in the ileum of 7 d broilers（P<0.05）and significantly reduced E.coli live bacteria count in the ileum of 3,7 and 14 d broilers（P<0.05）.TBNE significantly increased the concentration of butyric acid in the ileal of broilers on 3 d（P<0.05）,with an optimal dose of4 mg/egg for TBNE.It was evident that in ovo feeding TBNE could promote the growth and development of various intestinal segments in the small intestine of broilers,improve the efficiency of nutrient absorption and regulate the intestinal microenvironment,thus promoting the early growth and development of broilers.LPS tapping on 7 d broilers in the control and 4 mg/egg test groups revealed that TBNE significantly reduced the negative effects of LPS on mean body weight,ADFI,ADG,VH,CD,VH/CD（P<0.05）.The ileal p H and the Lactobacillus live bacteria count in the 4 mg/LPS group were significantly higher than LPS group（P<0.05）,the E.coli live bacteria count and butyric acid concentration were significantly lower than that of LPS group（P<0.05）.Compared to the control,the gene expression of acyl coenzyme A synthase short chain family member 2（ACSS2）,P₂Y₁receptor（P₂Y₁）andβ-catenin was significantly higher（P<0.05）and the gene expression of interleukin 6（IL-6）was significantly lower（P<0.05）in the 4 mg/egg group.Gene expression of ACSS2,P₂Y₂ receptor（P₂Y₂）,interleukin 1β（IL-1β）,IL-6 and interleukin 10（IL-10）were significantly increased（P<0.05）and P₂Y₁,β-catenin and enoyl coenzyme A hydratase 1（EHC1）were significantly decreased（P<0.05）in LPS group than that of control.Compared to the control,gene expression of ACSS2,P₂Y₂ and IL-10 in the 4mg/LPS group was significantly increased（P<0.05）and P₂Y₁,β-catenin and EHC1 in the 4mg/LPS group was significantly decreased（P<0.05）.The gene expression of ACSS2,P₂Y₁,P₂Y₂,β-catenin and IL-10 in the 4 mg/LPS group was significantly increased（P<0.05）,and IL-6 was significantly decreased（P<0.05）compared to the control.The gene expressions of ACSS2,P₂Y₁,P₂Y₂,β-catenin and IL-10 were significantly higher（P<0.05）,and IL-1βand IL-6 were significantly lower（P<0.05）in the LPS group compared to the control.Therefore,in ovo feeding TBNE could promote broiler intestinal energy metabolism and regulate intestinal health.It can also resist the damage caused by LPS to the intestinal tract,effectively alleviate intestinal stress and maintain intestinal health by regulating intestinal energy metabolism and immune response.In summary,TBNE improved the water solubility and dispersibility of tributyrin,and the particle size of TBNE was reduced to less than 300 nm through process optimization.TBNE could resist the acidic environment and pepsin action during digestion and remain stable in the stomach.Then,TBNE were more easily decomposed and digested after entering the intestine,thus improving the bioavailability of tributyrin.In ovo feeding TBNE could promote the growth and development of broiler’s intestinal,improve the efficiency of nutrient absorption and regulate the intestinal microenvironment,thus promoting the early growth and development of broilers.The broilers with TBNE could regulate the expression of related genes（ACSS2,P₂Y₁,P₂Y₂,IL-10,β-catenin up-regulated,IL-1β,IL-6 down-regulated）,promote butyric acid metabolism and cell proliferation,maintain intestinal integrity,regulate immune response,resist damage caused by LPS in intestine,thus effectively alleviating intestinal stress and maintaining intestinal health.