Animals and experimental design
A total of 45 Junmu No.1 White piglets ( Sanjiang white Pig × Seghers hybrid ) with the average body weight of 4.62 ± 0.06 kg (14 d of age) were randomly assigned to 3 treatments with 3 pigs per pen and 5 replicate pens per treatment and weaned at 21 d. Piglets were purchased from the original breeding farm of Jilin University, Changchun City, Jilin Province. The control group (CG) fed the basal diets from 14 d to 24 d of age. The low-dose group (LG) and the high-dose group (HG) fed the basal diets supplemented with 40 or 80 mg/kg β-carotene from 14 d to 24 d of age, respectively. The basal diet was prepared according to the NRC (2012) nutritional requirements of piglets. The ingredients of the basal diets are shown in Table 1. All piglets were housed in a temperature-controlled nursery (32-34℃) and were offered ad libitum access to water and creep feed.
At 21 d and 24 d of age, the body weights of the piglets were recorded. At 24 d of age, all piglets were euthanatized by intravenously injection of pentobarbital sodium (50 mg/kg body weight) and bled via jugular venipuncture. The piglets abdominal walls were opened using a scalpel and the complete intestine was removed to observe the intestinal mucin. Approximately a 2 cm of at the midpoint of the duodenum, jejunum and ileum was collected, fixed in 4% paraformaldehyde and kept at 4 °C for Periodic Acid-Schiff (PAS) stain immunofluorescence. Section of the midpoint of the duodenum, jejunum and ileum were collected, rinsed with normal saline, blotted dry with filter paper, frozen in liquid nitrogen, and stored at -80 ℃ for real-time PCR.
The body weight (BW) of piglets and amount of the feed were recorded at 21 d and 24 d of age. The average daily feed intake (ADFI), average daily gain (ADG) and d feed conversion rate (FCR) were calculated during the experiment period.
ADFI [g/days] = Total consumption [g]/(Test days [day] × Total number of piglets).
ADG [g/day] = (Final BW [g] − Initial BW [g])/(Test days [day] × Total number of piglets)
FCR [%] = ADFI/ADG
Detection of immunoglobulins and inflammatory factors in serum
The blood samples were placed into RNAase free tubes and then left to stand at the room temperature for 30 minutes. The blood was centrifuged at 3000 rpm for 15 minutes and then the serum was placed in a 1.5 ml centrifuge tube for the test.
Immunoglobulins (IgA、IgG and IgM) and inflammatory factors (TNF-α, IL-6, IL-8 and IL-10) were measured using a pig ELISA quantification kit (Bethyl Laboratories, Montgomery, TX, USA) and ELISA starter accessory package (Bethyl Laboratories) according to the manufacturer’s instructions. The plates were read at 450 nm with a micro plate reader (Multiskan FC; Thermo Fisher Scientific, Waltham, MA, USA).
Intestinal tissue PAS stain
The 4% paraformaldehyde-fixed and paraffin-embedded sections of the duodenum, jejunum and ileum were used for PAS stain. Firstly, sections of 5-mm thickness were deparaffinized and rehydrated then treated with 0.5% periodate solution for 10 minutes. Secondly, the sections were rinsed with running water for 5 min. Thirdly, the sections were then incubated in Schiff reagent for 20 min in the dark then washed with sulfite rinse for 2min and rinsed with running water for 10 min. Finally, The sections were hematoxylin counterstaining, dehydration, transparent and neutral gum seal. Images of immunofluorescent sections were captured using an Eclipse Ti-SR microscope with a DS-U3 Image-Pro system (Nikon).
The 4% paraformaldehyde-fixed and paraffin-embedded sections of the duodenum, jejunum and ileum were used for immunofluorescence. Firstly, sections of 5-mm thickness were deparaffinized and rehydrated then processed for antigen retrieval. Secondly, the sections were incubated in hydrogen dioxide for 10 min in the dark. Then, the sections were incubated with primary antibody MUC1 (1:200 dilution). Cy3-conjugated Affinipure Goat Anti-Rabbit IgG(H+L) antibody was used as secondary antibody (1:50 dilution). Nuclei were stained with Hochest (Solarbio, Beijing). Images of immunofluorescent sections were captured using an Eclipse Ti-SR microscope with a DS-U3 Image-Pro system (Nikon). MUC1 antibody purchased from Biorbyt. Secondary antibody purchased from Proteintech.
Analysis of mRNA abundance by real-time PCR
Total RNA was extracted from jejunum tissue using RNAiso Plus (TaKaRa Code:9109). The yield and purity of the RNA were evaluated using a NanoDrop 2000 (Thermo Scientific). RNA (1 μg) was used to generate cDNA (PrimeScriptTM RT reagent kit with gDNA Eraser, TaKaRa Cat# RR047A) in a volume of 20 μL. Real-time polymerase chain reaction was performed in a total volume of 20 μL using SYBR® Premix Ex TapTM II (TaKaRa Cat# RR820A). The primer sets used were designed with software ( Sangon Biotech Co., Ltd, Shanghai) and the sequences are listed in Table 2. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and β-actin were used as housekeeping genes in this study. The relative mRNA expression of the target genes was determined using the 2–∆∆CT method .
SPSS (SPSS GmbH, Munich, Germany) version 17.0 was used for data analysis. The statistical differences between groups were analyzed via a one-way ANOVA followed by a post-hoc least significant difference test. The results are given as the means ± SEM. Statements of statistical significance were based on P ≤ 0.05.