All experimental protocols were approved by the Animal Care and Use Committee of the shanghai Jiaotong University. The study took place at the experimental study farm of a feeding company (xinnong feed ltd).
Animal, diet and animal management
Twelve second-parity sows (hybrid Topigs 20 breed sows, Dutch Landrace × Great York) and their piglets [(Dutch Landrace × Great York) × Duroc] were used in the experiment. On day 85 of gestation, sows were equally divided into two groups, with six replicates per group and one sow per replicate. The back fat thickness of sows were measured at day 84 of gestation, and 12 sows which had similar back fat thickness (Soybean oil group: 15.50 ± 0.61 vs Fish oil group: 14.83 ± 0.79 mm; P = 0.52) were selected for our study. The back fat thickness was measured at the level of the last rib on each side and 65 mm from the midline by using the digital back fat indicator (BQT-521, Renco Lean-meater, USA).
From the 84th day of gestation until the 16th day of lactation, all 12 sows were divided into two dietary treatment groups: the first group was fed the soybean oil maternal diet (SD) and the second group was fed the fish oil supplemented diet (FD) during the experimental period. Diets were formulated according to the sow’s nutrient requirements from National Research Council (NRC, 2012) [9]. For gestation diet, the SD was composed of 3% of soybean oil to make the n-6: n-3PUFA ratio 8.8:1, while the FD was composed of 0.5% of soybean oil + 2.5% of fish oil to make the n-6: n-3PUFA ratio 1.6:1; For lactation diet, the SD was composed of 3.5% of soybean oil to make the n-6: n-3PUFA ratio 10:1, while the FD was composed of 0.7% of soybean oil + 2.8% of fish oil to make n-6: n-3PUFA ratio 2:1. Diet formulations are shown in Supplementary Table 1. The fatty acid composition in diets was determined as described by Raes et al.[10],and the fatty acid composition for experimental diets was shown in Table 1.All diets were mash feed and were stored in vacuum dark storage bags per 20 kg, and kept in 24-28℃ constant temperature warehouse before using.
From the 85th day to the 109th day of gestation, all pregnant sows were housed individually in gestation crates (2.1 × 0.65 m) and were fed gestation diet. The gestational sows were fed with 3.0 kg (3.0 kg /day diet was limited for sows during the late gestation period, so sows can eat up diets for each day ) and supplied twice per day (06:00 and 13:00)from the 84th day of gestation to 5 day before farrowing. From the 110th day of gestation to farrowing day, feed allowance was decreased by 0.5kg/day until no feed was supplied. On the 2nd day after farrowing, the sows were supplied diets 3 times per day (06:00, 13:00 and 18:00) with 0.75kg/day initially, and diet was then increased gradually by 0.75kg/day until reaching ad libitum. The room temperature of the gestation and farrowing units was approximately 24–28℃. Sows freely got access to water during the entire experiment.
All suckling piglets also were housed in corresponding farrowing unit with incubator and heat lamp for piglets from farrowing day to weaning day. During 48 h post-farrowing, the litter size was equalized to achieve 10–13 pigs by the means of cross-fostering within the same treatment group according to the number of effective nipples in each sow. Piglets were not fed the standard creep feed before 16 d. Piglets freely got access to water.
LPS treatment on piglets
On the 14th day after birth, twelve piglets (3 males and females in the SD group and the FD group) were selected in the study. One piglet per litter was selected and the average body weight is similar between the SD group and the FD group (SD: 4.31 ± 0.04 vs FD: 4.18 ± 0.09 kg; P = 0.23). Blood sample (5mL) were collected from each selected piglet, and then all selected piglets were administrated the cervical side behind the left ear with E. coli LPS at 80μg/kg BW. The LPS (Escherichia coli serotype 055: B5, Sigma Chemical, St. Louis, MO 63103, USA) was dissolved in sterile 0.9% NaCl solution (500 mg LPS per liter of saline). Blood samples (5mL) were collected from each pig at 5 h and 48h post-LPS challenge. All piglets were slaughter at 48h post-LPS challenge.The internal organs (intestine, liver, kidney, spleen, heart and pancreas) of the piglets obtained immediately after slaughtering and measured to calculate the relative organ weight and length to the body weight.
Blood and tissue sample collection
Blood samples. Blood samples from the sows were collected from the auricular vein. Each piglet was anaesthetized with an intramuscular neck injection of pentobarbital sodium (35mg/kg BW) and blood sample was then collected from the front cavity vein of each piglet. All blood samples were kept in heparinized tubes and centrifuged at 2550 × g for 10 min at 4°C. The supernatant fraction was divided and stored at −20 °C for subsequent analysis.
Liver samples of piglets. The posterior half of liver samples were obtained immediately after slaughtering. The livers were washed in physiological saline, collected into 5 ml freezing tubes, frozen in liquid N2, and then stored at −80°C.
Analysis of oxidant and antioxidant content
The analysis of malondialdehyde (MDA) (Cat No. A003-1-2), total superoxide dismutase (T-SOD) (Cat No. A001-1-2), glutathione peroxidase (GSH-Px) (Cat No. A005-1-2) and total antioxidant capacity (T-AOC) (Cat No. A015-3-1) in plasma was determined according to the manufacturer’s instructions (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). All absorbance levels were determined with a microplate reader (Synergy 2, BioTek, USA).
Analysis of cytokines in Plasma
The concentration of cytokines including interleukin-1β (IL-1β), interleukin-6 (IL-6), TNF-α and interleukin-10 (IL-10) was determined using porcine- specific ELISA kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer’s instructions. Absorbance values were read in a 96-well plate reader (Synergy 2, BioTek, USA) at 450 nm. A four parameter logistic curve-fit was generated using ELISA Calc software v0.1 (Comple-Software. Iowa City, IA). The concentration of cytokines in the plasma was calculated by comparison with a standard curve.
Quantitative real-time PCR
Total RNA was isolated from liver samples using Total RNA Kit Ⅰ (50) (Cat no. R6834-01; OMEGA, USA). RNA quality was verified by both agarose gel (1 %) electrophoresis and spectrometry (A260/A280, Beckman DU-800; Beckman Coulter, Inc.). One μg of RNA was reverse transcribed using the PrimescriptTM RT Reagent Kit with gDNA Eraser (Perfect Real Time) (RR047A, TaKaRa, Japan). Primers for all target genes are shown in Supplementary Table 2. Quantitative real-time PCR (RT-qPCR) was used to determine the relative expression level of target genes using the one-step SYBR® Premix Ex Taq (TLi RNaseH Plus) (RR420A; TaKaRa, Japan). Briefly, the final volume of the reaction mixtures (20 μL) contained 10 μL of SYBR Premix Ex Taq (Tli RNaseH Plus), 0.8μL of the primer pair, 0.4μL of ROX Reference Dye Ⅱ, 2 μL of cDNA and 6.8 μL of sterile water. β-actin was used as the endogenous control gene to normalize the expression of target genes. The relative quantification of gene amplification by RT-qPCR was performed using the value of the threshold cycle (Ct). The comparative Ct value method using the formula 2-ΔΔCt was employed to quantify the expression levels of target genes relative to those of β-actin using the following formula:
2-ΔΔCt (∆∆Ct = (Cttarget gene– Ctβ-actin) treatment – (Ct target gene– Ct β-actin) control)[11]
Western Blot Analysis
The proteins in liver samples were extracted and mixed with loading buffer as previously described by Luo et al. [7]. Forty μg of proteins were separated on 10% SDS-PAGE gels and electro-transferred to polyvinylidene difluoride (PVDF) membranes (0.45μm pore size, IPVH00010, Millipore, MA). The membranes were blocked for 2 h with 5% (w/v) skimmed milk powder (Cat No.D8340, Solarbio, Shanghai, China) in tris-buffered saline-tween (TBS-T) [0.5M NaCl (S7653, Sigma-Aldrich, Shanghai, China), 20mM Tris (Amresco, Shanghai, China), pH 7.5, and 0.1% (v/v) Tween-20 (P7949, Sigma-Aldrich, Shanghai, China)], then washed three times with TBS-T, and incubated overnight at 4℃ with primary antibodies following dilutions in 5% skimmed milk powder or BSA (Cat No.0218054950, ChromatoPurTM, New Zealand).The primary antibodies were anti-JNK (1:200, sc-571, Santa Cruz, USA), anti-p-JNK(1:500, orb10951, Biorbyt Ltd, UK),anti-p38α (1:2000, sc-535, SantaCruz, USA), anti-p-p38 (1:200, sc-7973, Santa Cruz, USA), anti-ERK1/2 (1:1000, number 9102, Cell Signaling Technology, USA), anti-phospho-ERK1/2 (1:2000, number 4370, Cell Signaling Technology, USA), anti-IκBα(1:1000, number4814, Cell Signaling Technology, USA) and anti-p-IκBα (1:1000, number 9246, Cell Signaling Technology, USA) antibodies. After washing three times with TBS-T, the membranes were then incubated with goat anti-rabbit (1:10000, ab97051, Abcam, UK) or goat anti-mouse IgG-HRP (1:2000, sc-2005, Santa Cruz, USA) antibodies for 2 h. Afterwards, blots were developed using AmershamTM ECLTM Prime Western Blotting Detection Reagent (Catalogue No.RPN2232, Healthcare UK limited little Chalfont). Image acquisition was performed on an enhanced chemiluminescence detection system (Tanon, Shanghai, China). Image J software was used to quantify the density of the specific protein bands.
Statistical analyses
All variables were tested for normal distribution by Shapiro–Wilk test. Individual sow or piglet was the experimental unit for the indices. The data were analysed by using the procedure of t-test (IBM SPSS Statistics 20).Because of the differences among treatments at the start of piglet study, plasma TG,IL-1β,IL-6 and IL-10 concentrations of piglet before LPS challenged were regarded as a covariate. Results were expressed as means and the standard error of the mean (SEM). A P-value less than 0.05 was considered to be statistically significant.