Animals and experimental design
This study was conducted in Guangzhou DaBeiNong Agri-animal Huabandry Science and Technology Co., Ltd. A total of 84 Landrace × York sows (parities 2–5) with similar farrowing time were allocated to four dietary treatment groups with each sow as a replicate in a completely randomized design using body weight at 85 day of gestation as a block (n=21 per treatment). The sows in the control group (CON) received a basal gestation or lactation diet without added CS from 85 day of gestation to 21 day of lactation, while the sows in the CS group were fed a basal diet supplemented with 100, 300, or 500 mg/kg of CS (CS100, CS300, and CS500 diet). All diets were formulated to meet the National Research Council (NRC, 2012) requirements of nutrient standards for gestational and lactational sows. The ingredients and compositions of the basal diet are shown in Supplementary Table S1.
Sows were housed in individual stalls and fed twice (07:30 and 17:00) a day with a constant amount of 3 kg during late gestation. During the entire lactation period of 21 days, the piglets had no access to the sow’s feed or to creep feed. Sows and piglets were given free access to water throughout the experiment. On the day of farrowing, sows were offered 2.0 kg of the lactation diet, followed by increasing the amount to 3.0 kg daily until ad libitum feeding. The reasons for the sows eliminated from this study were recorded in detail and shown in Supplementary Table S2.
Measurements of reproductive performance and sample collection
After farrowing, the number and weight of the piglets born, born alive, stillbirths and mummies were recorded, and invalid piglets included stillbirth and mummy. Piglets were weaned at 21 day of lactation.
During sow farrowing, umbilical cords were tied with a short silk line and each piglet was marked with a numbered tag to match the individual piglets with their placentae. After placental expulsion and weight recording, the placentae were collected and snap-frozen in liquid nitrogen (3 to 4 cm from the cord insertion point), and the other fresh placental tissues were immediately fixed in 4% paraformaldehyde. Placental efficiency was calculated by dividing piglet weight by placental weight [17]. In this study, the mean birth weight of the 1181 piglets was 1.4 ± 0.22 kg (mean ± standard error). The placentae were assigned to two categories according to piglet birth weight: <1.0 kg (low birth weight, LBW) and 1.4-1.6 kg (normal birth weight, NBW). Litter size at birth was also categorized into 2 classes based on the average number of piglets born alive per litter (15.0 piglets): low yield sow (the number of born alive piglets < 15.0) and high yield sow ( the number of born alive piglets ≥ 15.0).
Sow blood samples (n=8 per group) were collected from the ear vein of the fasted gilts using 10 mL centrifuge tubes at farrowing and at weaning and centrifuged at 3,000 × g and 4°C for 15 min to recover the serum. The blood samples of piglets were collected from the anterior vena cava of the piglet whose body weight was closest to the average body weight of the litter at birth (NBW piglets, n=6) on the parturition day and centrifuged at 3,000 × g and 4°C for 15 min to recover the serum. At 0.5 h before the birth of the first piglet, colostrum (20 mL) was collected from the functional glands of each sow. Finally, these samples were stored at -80 ℃ until further analysis of the oxidative parameters.
Oxidative stress parameters in serum, colostrum and placenta
The total antioxidant capacity (T-AOC), glutathione (GSH), and malondialdehyde (MDA) were determined using the commercial kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer's procedures.
Total protein concentrations in placenta and colostrum were measured according to the instructions of the bicinchoninic acid protein assay kit (Beyotime, Beijing, China). T-AOC, GSH, and MDA in placenta and colostrum were normalized to the total protein. T-AOC is associated with the elimination of free radicals and ROS, blocking peroxidation and thus preventing lipid peroxidation and removing catalytic metal ions, while MDA is the end product of lipid peroxidation and an excellent indicator of oxidative stress [18].
Placental vascular density
Placental tissues fixed in 4% paraformaldehyde were embedded in paraffin and sectioned at 5 μm thickness, followed by staining with hematoxylin and eosin (H&E). The placental vessels in these areas were also traced using a projecting microscope (Olympus CX41, Japan) and then quantified as previously described [10].
Cell culture and treatments
Porcine vascular endothelial cells (PVECs) were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and cultured in 1640 medium with 10% fetal bovine serum (FPS), 100 U/mL penicillin, and 100μg/mL streptomycin at 37°C in 5 % CO2 atmosphere. The PVECs with cobblestone morphology were passaged at 90% confluence and used for experiments within five passages. Subsequently, the cells were seeded at varying densities incubated for 24 h, and then treated with CS or H2O2 for the desired time periods. For some experiments, cells were pretreated with various concentrations of CS (0.5, 1 or 2mM) for 2 h and/or then treated with various concentrations of H2O2 (100, 200 or 300μM) for 24 h.
Cell viability assay
The PVECls (10,000 cells per well) were seeded in 96-well plates and incubated for 24 h. After different treatments, cell viability was measured using cell counting kit-8 assay (CCK-8) (Beyotime) as instructed by the manufacturer. The absorbance of each well at 490 nm was measured using a microplate reader (Bio-Rad Laboratories, Hercules, CA).
Measurement of intracellular ROS generation
The accumulation of intracellular ROS was examined using the ROS assay kit (Beyotime) according to the manufacturer’s instructions. Briefly, cells were grown in a 96-well plate and subjected to different treatments. After incubation with 10 μM 2,7-dichlorofluorescein diacetate at 37 °C for 20 min, the fluorescence intensity of the the cells was measured using the fluorescence plate reader (BD Falcon, Bedford, MA, USA) at Ex./Em. = 488/525 nm.
Scratch healing assay
The wound healing scratch assay was used to assess cell migration as previously described [19]. Briefly, cells were seeded onto a 6-well plate and cultured overnight until the formation of a confluent monolayer, followed by making a scratch wound with a 200-µL pipette tip and measuring the effects of H2O2, CS, and stattic on scratch healing at 24 h after the scratch. The images of the wounded areas were captured using an Olympus inverted microscope and quantified using the ImageJ software.
In vitro tube formation assay
Matrigel tube formation assays were used to assess the in vitro angiogenic activity of PVECs. Briefly, after experimental treatment, PVECs were seeded in 96-well plates precoated with 50 μL Matrigel (BD company, USA) at a density of 1 × 104 cells per well. After 4 h incubation, Matrigel-induced morphological changes in HRGECs and their tubular networks were photographed at 50 or 100× magnification for analysis using Image J software.
Trans-well migrationassay
The chemotactic migration of PVECs was assayed using a trans-well chamber equipped with a polycarbonate filter with a diameter of 6.5 mm and a pore size of 8 µm. Briefly, after different treaments, PVECs were suspended in 1640 medium to a final concentration of 5 × 104 cells/ml and were placed in the upper wells of the chamber, while the lower chamber was filled with 600 μl medium containing 10% FPS. After incubation at 37°C for 48 h, the cell culture inserts were collected. The cells on the upper side of the filters were removed with cotton-tipped swabs, while the cells on the underside of the filters were fixed with 4% formaldehyde for 30 min, which were stained with crystal violet for 20 min and counted in five randomly chosen fields.
Quantitative real-time RT-PCR (qRT-PCR) analysis
Total RNA from placenta or PVECs was extracted with the reagent box of Total RNA Kit according to the manufacturer’s instructions. The concentration of RNA was quantified using a NanoDrop® 2000 (Thermo Fisher, USA). After reverse transcription using Primer Script TM RT reagent Kit (Takara, Qingdao, China), quantitative PCR (qPCR) was conducted using SYBR Green on a QuantStudio 6 RealTime PCR System (Thermo Fisher, USA) under the conditions of denaturation at 95 ◦C for 10 min, amplification at 95 ◦C for 15 s and 60 ◦C for 1 min for 40 cycles. Each target gene was individually normalized to the reference gene β-actin by using the quantification method of 2−ΔΔct. Primers used in this study are shown in Supplemental Table S3.
Western blotting
Total proteins were extracted from PVECs using the protein extraction kit (Beyotime, Beijing, China) according to the manufacturer’s guide. Briefly, an amount of 10 μg protein was loaded and separated by SDS–PAGE gel electrophoresis, followed by transferring the proteins onto the polyvinylidenedi fluoride membranes (Merck Millipore). After blocking with TBS/T buffer containing 5% milk, the membranes were incubated with the primary antibodies against vascular endothelial growth factor A (VEGF-A) (19003-1-AP, Proteintech, USA, 1:1000), NADPH oxidase 2 (NOX2) (19013-1-AP, Proteintech, USA, 1:1000), signal transducer and activator of transcription-3 (Stat3)(ab76315, Abcam, USA, 1:1500), p-Stat3 (ab68153, Abcam, USA, 1:1500), and β-actin (4970, CST, USA, 1:1000). Subsequently, the membranes were incubated with appropriate HRP-conjugated anti-rabbit IgG secondary antibody (AS014, Abclonal, China, 1:5000). Images were captured using the ChemiDoc MP system (Bio-Rad, Hercules, CA, USA), and band densities were quantified using Image Lab software (Bio-Rad, Hercules, CA, USA) and then normalized to β-actin content.
Immunofluorescence
Placental tissues fixed in 4% paraformaldehyde were embedded in paraffin and sectioned at 5 μm thickness for platelet endothelial cell adhesion molecule-1 (CD31) immunofluorescence as described previously [19].
Statistical analysis
Data are presented as mean ± SEM and were statistically analyzed using one-way ANOVA and Duncan’s multiple-range test in SPSS 20.0 (SPPS Inc., Chicago). Tamhane’s T2 test was used to assess variance heterogeneity. The stillbirth, LBW, and invalid rates were analyzed using the Chi-square test. Pearson’s correlation coefficient was used to analyze the correlation between piglet birth weight and placental vascular density, T-AOC, and MDA. Additionally, polynomial contrasts were used to evaluate the linear and quadratic effects of CS supplementation on the various parameters measured in the sow experiment. Differences were considered significant at P < 0.05, and a tendency was considered at 0.05 ≤ P < 0.1.