| Aronia melanocarpa pomace(AMP)is the main by-product produced during the deep processing of its fruit,which is rich in natural polyphenols antioxidant,such as anthocyanin,chlorogenic acid,quercetin and so on.It has great potential to add AMP as an unconventional feed resource to the feed of growing and finishing pigs.In this experiment,27 60-day-old pigs were divided into three groups according to the principle of similar body weight.The control group(CON)was fed with basic diet,and the experimental group was fed with 4 % and 8 % additional AMP in the basic diet respectively.Three replicates were set in each experimental group,and three pigs in each replicate were fed for 7 weeks to detect their effects on growth index,intestinal development and intestinal flora.To evaluate the application value of AMP as an unconventional feed resource in pig production.At the same time,in order to further develop the active components of natural polyphenols in AMP,after consulting and analyzing the polyphenols in AMP,it was found that Procyanidin B2(Procyanidin B2,PCB2)was the polyphenol component with the highest content,and judging from its biological function,PCB2 may also play an important role in this experiment,therefore,the second part of the in vitro experiment carried out different concentrations of PCB2 on porcine intestinal epithelial(IPEC-J2)cells,to explore its effects on IPEC-J2 cell apoptosis,inflammation,tight junction related genes and proteins,and to explore the feasibility of PCB2 as a feed additive in vitro.The conclusions of the study are as follows:(1)The results of feeding experiment showed that the addition of 4 % AMP significantly increased the average daily gain(ADG)of pigs(P < 0.05),and the addition of 4 % and 8% AMP had a tendency to improve the average daily feed intake(ADFI)and the ratio of feed to meat(F/G),the blood biochemical indexes of pigs were in the normal range,the addition of 4 % AMP in feed significantly increased the total antioxidant capacity(T-AOC)of jejunum and significantly decreased the content of malondialdehyde(MDA).In terms of intestinal development(P < 0.05),4 % AMP significantly increased the villus length and villus length / crypt depth of pig jejunum(P < 0.05),and different levels of AMP had no significant effect on the pig cecum morphology.In terms of intestinal flora,diets supplemented with different levels of AMP had no significant effect on the relative abundance of Firmicutes and Bacteroidetes in the intestinal contents of pigs,while the relative abundance of Proteobacteria decreased significantly(P < 0.05).At the genus level,the relative abundance of Lachnospira,Solobacterium,Prevotella,Prevotella＿9,Romboutsia and Butyrivibrio was significantly increased by adding 4 % AMP(P < 0.05),different AMP supplementation significantly decreased the relative abundance of Escherichia-Shigella and Pseudoscardovia(P < 0.05).After the detection of intestinal volatile fatty acids,it was found that the addition of 4% AMP significantly increased the contents of acetic acid and butyric acid in pig intestinal contents(P < 0.05),the addition of 8 % AMP significantly increased the contents of propionic acid,butyric acid and caproic acid(P < 0.05).In addition,different levels of AMP significantly decreased the gene expression levels of tumor necrosis factor α(TNF-α),interleukin 6(IL-6)and interleukin 8(IL-8)in pig jejunum(P < 0.05),and significantly increased the gene expression levels of tight junction protein 1(ZO-1)and blocking protein 1(Claudin-1)in intestine(P < 0.05).However,the addition of 8% AMP to the feed had no significant effect on the gene expression levels of interleukin-1β(IL-1β)and blocking protein(Occludin).To sum up,under the conditions of this experiment,adding 4 % AMP to the basic feed is more beneficial to the overall health and growth of pigs.The results of this study provide a theoretical basis for the application of AMP as an unconventional feed raw material in pig breeding.(2)The results of in vitro experiment showed that IPEC-J2 cells treated with 40μM PCB2 for 24 h could significantly increase the cell viability(P < 0.05),while 20,40 and 60 μM PCB2 had no significant effect on the content of lactate dehydrogenase(LDH)in the culture medium.Through the detection of cellular reactive oxygen species(ROS),it was found that 20,40 and 60 μM PCB2 had no significant effect on the ROS level of IPEC-J2 cells.By further detecting the expression level of inflammatory related factors,it was found that 20 μM and 40 μM PCB2 significantly decreased the gene expression levels of pro-inflammatory cytokines IL-1β and TNF-αin IPEC-J2 cells(P < 0.05),while different concentrations of PCB2 had no significant effect on the gene expression levels of IL-6,IL-8 and IL-10.Real-time fluorescence quantitative PCR(RT-q PCR)and Western Blot were used to detect apoptosis-related genes and proteins.It was found that 20,40 and 60 μM PCB2 treatment had no significant effect on the gene expression and protein content of B lymphocyte tumor-2associated X protein(BAX)and B lymphocyte tumor-2(BCL-2),but 20 μM PCB2 treatment significantly decreased the rate of apoptosis(P < 0.05).In terms of intestinal barrier related gene expression,compared with the control group,20,40 and60 μM PCB2 treatment had no significant effect on the gene expression and protein content of Occludin and Claudin-1,but 40 μM PCB2 treatment significantly increased ZO-1 gene expression and protein content in cells(P < 0.05).Based on the experimental results,it was found that normal IPEC-J2 cells treated with 40 μM PCB2 could improve the ability of anti-inflammation and anti-apoptosis and promote the expression of tight junction protein,indicating that PCB2 can be used as a potential feed additive in animal breeding.To sum up,it is safe for AMP to be added to the feed of growing and finishing pigs as an unconventional feed resource.under the conditions of this experiment,the addition of 4 % AMP in feed is more beneficial to the overall health and growth of pigs.At the same time,normal IPEC-J2 cells treated with 40 μM PCB2 can improve the ability of anti-inflammation and anti-apoptosis and promote the expression of tight junction protein,indicating that PCB2 can be used as a potential feed additive in animal breeding. |
