The diets of sows with sh oil might decrease the oxidative stress and inammatory response in sows, but increase the susceptibility to inammatory response in their offspring

Backgroup: This aim of this study was to investigate that the effect of supplement maternal diet with sh oil on the oxidative stress and inammatory response of sows and their offspring. Methods: Twelve sows were divided into two groups. Sows were fed soybean oil diet (SD) or soybean oil + sh oil diet (FD) from gestating to lactating period. The blood samples of lactating sows were collected. At the age of 14 days, one piglet was selected from each litter. After the blood was collected from the anterior vena cava, LPS was injected into the neck muscle. The blood was collected 5 hours after LPS injection. At 48h after LPS injection, blood and liver samples were collected. Results: Maternal sh oil supplementation improved the health of sows by increasing the level of HDL-C and decreasing the levels of AKP and TNF-α in the plasma of sow (P<0.05), and induced a positive effect on litter immune status associated with a modication of both IgG in the colostrum and IL-10 in the milk(P<0.05).In addition, antioxidant capacity in piglets increased by improving the level of GSH-Px and T-AOC (P<0.05) in the plasma of piglets 48h after LPS challenged. Meanwhile, the expression of GSH-Px mRNA and p-ERK protein in the livers of piglets was inhibited (P<0.05). However, in FD group, the level of IL-1β and IL-6 in the plasma of piglets were signicantly higher before and after LPS challenged (P<0.05). The expression of NF-κB mRNA and p-IκB-α protein was increased in the liver of piglets (P<0.05). Conclusion: These results indicated that the diet of sow with sh oil might decrease the oxidative stress and inammatory response in sows and enhance the antioxidative ability in their progenies, but might increase the susceptibility to inammatory response in progenies. blots


Background
Neonatal piglet survival always bothered the modern pig industry, because the imperfect immune system of neonatal piglet makes it is sensitive to resist pathogen. The liver plays a central role in the regulation of lipid metabolism and is critical for host defense against invading pathogen and tissue repair in severe infection. Nutrition during pregnancy and lactation period may have a fundamental impact on fetal immune development [1,2]. Fish oil is rich in long chain n-3 polyunsaturated fatty acids (n-3 LC-PUFAs), such as 20:5n-3 (EPA) and 22:6n-3 (DHA), which are essential fatty acid for pig and exert bene cial anti-in ammatory effects in animal and cell models [3]. The sow's milk were the main source of nutrient for the sucking piglets. The lactating diet of sow with 3 ∼ 5% sh oil is bene cial for the growth of sucking piglets [4][5][6]. Therefore, maternal nutrition is closely related to the health of the progeny.
Weaning is abruptly stressful in the neonates' life, and that stress can result in growth retardation and susceptibility to diseases in mammals.
Weaning stress increased oxidative stress and transaminase in the liver of piglets and induced apoptosis, which may be related to MAPK signaling pathway activated by weaning piglets [7]. Recent evidences from animal models indicate that LPS-induced acute liver injury is caused by a variety of factors including in ammation, oxidative stress and lipid metabolism disorders. In our previous study, the supplementation of sh oil in sow diet was associated with longer gestation, alleviate oxidative stress in sows on farrowing day and modulate in ammatory response in sows and their offspring [8]. As mentioned above, we hypothesized that the maternal diet with sh oil could change the in ammatory response of the sows and potentiate the resistance for in ammation in the piglets suckling sh oil supplemented sow under the stimulation of immune stress.
The objective of this study was to investigate that the effects the maternal diet with sh oil on the oxidative stress and in ammatory response of sows, as well as on the oxidative stress and in ammatory response in their offspring before and after LPS challenge.

Materials And Methods
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 84 th day of gestation until the 16 th day of lactation, all 12 sows were divided into two dietary treatment groups: the rst group was fed the soybean oil maternal diet (SD) and the second group was fed the sh 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 sh 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 sh 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 85 th day to the 109 th 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 84 th day of gestation to 5 day before farrowing. From the 110 th day of gestation to farrowing day, feed allowance was decreased by 0.5kg/day until no feed was supplied. On the 2 nd 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 14 th 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 N 2 , and then stored at −80°C.
Absorbance values were read in a 96-well plate reader (Synergy 2, BioTek, USA) at 450 nm. A four parameter logistic curve-t 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 (50) (Cat no. R6834-01; OMEGA, USA). RNA quality was veri ed 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 Primescript TM 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 onestep SYBR ® Premix Ex Taq (TLi RNaseH Plus) (RR420A; TaKaRa, Japan). Brie y, the nal 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 , 2 μL of cDNA and 6.8 μL of sterile water. βactin was used as the endogenous control gene to normalize the expression of target genes. The relative quanti cation of gene ampli cation by RT-qPCR was performed using the value of the threshold cycle (C t ). The comparative C t 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 (∆∆C t = (C ttarget gene -C tβ-actin ) treatment -(C t target gene -C t 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 di uoride (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- Image acquisition was performed on an enhanced chemiluminescence detection system (Tanon, Shanghai, China). Image J software was used to quantify the density of the speci c 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.

Organ indices of piglets
The organ indices of piglets were shown in Table 2. There were no differences in the relative intestinal length of piglets between the SD group and the FD group. The FD had no effect on the relative liver weight, pancreas weight, brain weight, spleen weight and kidney weight of piglets.
Effect of sh oil maternal diet on the lipid pro le and liver function of sows The FD treatment had signi cant increased plasma level of HDL-C compared with the SD treatment (P < 0.05) ( Table 3). There were no difference between the two groups for the level of TG, T-CHO and LDL-C in the plasma of sow. The level of AKP in the plasma of sow was lower in the FD group relative to the SD group on the 16 day of lactation (P <0.05). The FD had no signi cant in uence on the level of AST and ALT in the plasma of sows.
Effect of maternal diet with sh oil on the oxidative stress parameters and immune cytokines in the plasma of sows The FD decreased the concentration of TNF-α (P < 0.05), but had no signi cant in uence on the concentration of IL-1β, IL-6 and IL-10 in the plasma of sow on the 16 day of lactation (Table 4). Moreover, The FD had no signi cant in uence on the 8-iso-PG, T-SOD, GSH-Px and T-AOC in sow plasma on the 16 day of lactation (P > 0.05).
Effect of the maternal diet with sh oil on the content of chemical composition, immunoglobulins and immune cytokines in the colostrum and milk The FD signi cantly increased the content of IgG in the colostrum, but had no signi cant in uence on the content of β-casein, sIgA and IgM in the colostrum and milk ( Table 5). The concentration of TNF-α in the milk of the FD group was lower than that of the SD group (P <0.05), while the concentration of IL-10 in the milk of the FD group was higher than that in the SD group (P <0.05). The FD had no effect on the IL-1β, IL-6, TNF-α and IL-10 in the colostrum.
Effect of the maternal diet with sh oil on the lipid pro le and liver function in the plasma of piglets pre-/post-LPS challenge The FD group had signi cantly decreased plasma level of TG compared with the SD group on the 14 day of suckling piglets before LPS challenged (P < 0.05), but had no effect on the level of T-CHO, LDL-C and HDL-C in the plasma of piglets pre-LPS challenge. The FD had no signi cant in uence on the level of AST, ALT and AKP in the plasma of the suckling piglets pre-LPS challenge.
The level of T-CHO and LDL-C in the plasma of the suckling piglets from the FD group was lower than that from SD group at 5h post-LPS challenge (P <0.05). Conversely, the level of ALT and AKP increased in the FD group relative to the SD group in the plasma of the piglets at 48h post-LPS challenge(P <0.05).

Effect of the maternal diet with sh oil on the oxidative stress status in piglets
The activities of GSH-Px and T-AOC in the plasma of suckling piglets were higher in the FD group than that in the SD group at 48h post-LPS challenge (P < 0.05) (Fig. 1c, 1d, respectively), while the FD had no effect on the 8-iso-PG and T-SOD in the plasma of suckling piglets pre-/post-LPS challenge (P > 0.05) (Fig. 1a, 1b,respectively). Conversely, the relative expression of GSH-Px mRNA in the livers of piglets of the FD group was lower than that of the SD group at 48h post-LPS challenge (P < 0.05) (Fig. 1e).
Effect of the maternal diet with sh oil on in ammatory response in piglets The FD increased the concentration of IL-1β, IL-6 and IL-10 in the plasma of piglets pre-LPS challenge (P<0.05) (Fig. 2b, 2c, 2d, respectively), while the FD had no effect on the concentration of TNF-α in the plasma of piglets pre-/post-LPS challenge. The concentration of IL-6 in the plasma of piglets was higher in the FD group than that in the SD group at 48h post-LPS challenge (P<0.05) (Fig.2c). The concentration of IL-1β in the plasma of piglets was signi cantly higher in the FD group than that in the SD group at both 5h and 48h post-LPS challenge (P<0.05) (Fig.2b). Meanwhile, the relative expression of IL-1β mRNA in the livers of piglets was also higher in the FD group than that in the SD group at 48h post-LPS challenge (P<0.05) (Fig.2f). There is no diet effect on the relative expression of IL-6 and IL-10 mRNA in the livers of piglets at 48h post-LPS challenge.
Effect of the maternal diet with sh oil on mechanism parameters in piglets The relative expression of G-protein coupled receptor 120 (GPR120) the livers of piglets was lower in the FD group than that in the SD group at 48h post-LPS challenge (P < 0.05) (Fig. 3a). Conversely, the relative expression of TGF-beta activated kinase 1 (TAK1) and NFκB mRNA in the livers of piglets was higher in the FD group than that in the SD group at 48h post-LPS challenge (P < 0.05) (Fig. 3a).
Fish oil supplementation in maternal diet signi cantly down-regulated the p-ERK/ERK protein in the livers of piglets at 48h post-LPS challenge (P < 0.05) (Fig. 3b). Conversely, the expression of p-IκBα/IκBα proteins in the livers of piglets was signi cantly up-regulated in the FD group at 48h post-LPS challenge (P < 0.05) (Fig. 3b).

Discussion
In this study, we investigated that the effects maternal diet with sh oil on the oxidative stress and in ammatory response of lactating sows, as well as the effects on oxidative stress and in ammatory response of their offspring before and after LPS stimulation.
The supplementation of sh oil in the diet of perinatal sows may help to reduce the in ammatory response and improve the liver function of lactating sows. Our results showed that maternal diet with sh oil could increase the plasma HDL-C level and decrease the plasma AKP and TNFα levels, but had no signi cant effect on the markers of oxidative stress and antioxidant enzymes in the plasma of sow. These results were consistent with the report of Papadopoulos et al. (2009),which reported that sh oil diet signi cantly decreased the levels of IL-6 and TNF-α in the serum of sow on the 3 rd and 8 th day of lactation [12]. Tanghe et al. (2013) reported that the perinatal sows' diet with sh oil had no effect on the oxidative stress state of sows [13]. However, the inconsistent reports found that dietary sh oil had no effect on the expression of genes related to cholesterol synthesis and absorption in the liver of lactation sows, but had minor effect on the hepatic lipid metabolism which had no effect on the milk production of sows [14]. Shen et al. (2015) suggested that the supplementation of olive oil in the maternal diets signi cantly reduced IL-1β and TNF-α in the milk, but increased the oxidative stress of sows in sh oil group [15]. The reasons for these inconsistency may be associated with the experimental design, diet storage (vacuum dark bag storage or common bag storage) and the dietary antioxidant supply [16].
Dietary supplementation with sh oil to sows induced a positive effect on litter immune status associated with a modi cation of both cytokines and immunoglobulins (IgG and IgM) in milk [17]. Dietary sh oil resulted in an increase in antibodies in both blood and colostrum from sows after vaccination, together with an increase in antibodies, leukocyte and IgG in the blood of piglet [18]. n-3PUFA supplementation could reduce the in ammatory stimulation of LPS on mammary gland and the levels of IL-6, IL-8, IL-1 β and TNF-α in milk [15,19].Our results showed that the level of IgG and IL-10 in colostrum were increased and TNF-α in normal milk were decreased by adding sh oil to the diets of gestating and lactating sows.
In our study, at the age of 14 days, the TG level in the plasma of sucking piglets was reduced, but the levels of ALT,AKP,IL-1β,IL-6 and IL-10 in the plasma of sucking piglets were increased in the FD group. ALT and AKP is positively correlated with the degree of abnormal liver tissue [20][21][22].
IL-10 is an anti-in ammatory regulatory cytokine, which could induce antibody production and improve anti-in ammatory ability [23]. IL-10 in milk can cross the intestinal barrier and affect thymic development in progeny [24].The increase of IL-10 in plasma of sucking piglets might be transfer from milk to piglets. However, the increase of IL-1β and IL-6 in the plasma of piglets suggested the increase of in ammatory response in the FD group. This is inconsistent with the reports of Leonard 2019), which found that providing sh oil diet to sows beginning 5-7 days before delivery and during lactation can increase leukocyte and lymphocyte phagocytosis in piglets at weaning, and reduce the acute physiological stress response in the pigs postweaning by attenuating the release of in ammatory cytokines (IL-1β, IL-6, and TNF-α) [25,26]. The reason for this inconsistency may be related to the feeding time period and the dose of sh oil. In the previous studies, the intake of sh oil for sows is very limited before delivery, because the feeding allowance of sows was gradually decreased from 5-7 days before delivery in actual pig production. So far, the experimental research about sh oil supplementation for more than 4 weeks were limited. Luo et al.
(2013) reported that the diet of sow with sh oil from 10 days before delivery can reduce the expression of in ammatory cytokines in skeletal muscle and promote the growth of suckling piglets, but increase the expression of in ammatory cytokines in the spleens of weaned piglets by continuously feeding sh oil diet to weaned piglets [27].
Maternal sh oil supplementation potentiated antioxidant capacity in suckled piglets. Our results showed that there were no difference on the oxidative stress state in the piglets suckling sh oil supplemented sows before LPS challenged. However, 48 hours after LPS challenged, the levels of GSH-Px and T-AOC in plasma of piglet were increased in FD group, while the oxidative stress marker products had no effect. Marianne et al. (2018) reported that supplementation of DHA in piglets can signi cantly reduce the oxidative stress response caused by lipid peroxidation [28]. Although there is no report about the effect of dietary sh oil on the mRNA expression of antioxidant enzyme in the livers of piglets, placental antioxidant system has a proper capability to compensate for the oxidative stress through increasing gene expression of antioxidant enzymes in placenta [8,29,30]. We postulated that hepatic antioxidant system of piglets may also have an adaptive response to oxidative stress. In addition, oxidative stress can induce ERK phosphorylation in hepatocytes, but DHA can regulate the activation of ERK1/2 in MAPK [31, 32]. The cytokines produced by in ammatory stimulation could cause a secondary attack on the tissue by inducing ROS production, but DHA can directly reduce ROS produced by cytokines through inhibiting the activation of ERK1/2 [32] . In our study, the expression of GSH-Px mRNA and the phosphorylation of ERK protein in the liver of piglets were decreased in piglets suckling sh oil supplemented sows.
Interestingly, our results were inconsistent that the effects of sh oil supplementation on in ammatory response and oxidative stress in piglets suckling sh oil supplemented sows. The initial phase of the acute in ammatory response is characterized by the production of proin ammatory mediators followed by a second phase in which lipid mediators with pro-resolution activities may be generated [33]. Dysregulation of the dynamic in ammatory process will directly lead to tissue damage. LXA4, is an eicosanoid generated, which is one of the endogenous local mediators of resolution during acute in ammatory responses. NF-κB activation associated with both the onset and the resolution of in ammation in both rat carrageenin pleurisy and mouse carrageenan air pouch [34]. NF-κB DNA binding activity further increased at 24 and 48 h after in ammatory stimulation in the presence of IκBα protein [34]. Myeloid cells represent a major target for LXA4 and re ect the attenuation of NF-κB activity [35][36][37]. Therefore, n-6 PUFA participates in the resolution of in ammation [38]. In addition, the production of LXA4 requires the function of ALOX15 enzyme. When the same amount of n-3PUFA and n-6PUFA substrates exist, the enzyme shows the priority of using DHA and EPA instead of 20:4n-6 [39]. Our previous study reported that the total proportion of n-3PUFA in the livers of piglets suckling sh oil supplemented sows were higher than that of n-6 PUFA[40]. In our present study, the relative expression of NF-κB mRNA and the expression of p-IκBα/IκBα proteins in the livers of piglets after 48h LPS challenge were decreased. These results indicated that the continuous supplementation of sh oil for more than 4 weeks may affect the process of resolution of in ammation in the liver of piglets and lead to liver injury.

Conclusion
In summary, the diet of sow with sh oil might decrease the oxidative stress and in ammatory response in sows and enhance the antioxidative ability in their progenies, but might increase the susceptibility to in ammatory response in progenies.  SD=Soybean oil diet; FD=Fish oil diet; n. d=not detectable; n-3 = n-3 poly unsaturated fatty acid; n-6 = n-6 ploy unsaturated fatty acid; n-6: n-3=n-6 polyunsaturated fatty acids: n-3 polyunsaturated fatty acids.   All results are presented as mean ± SEM (n=6), SEM=pool SEM.

Abbreviations
*Mean values were significantly different between the two groups at the same time point P < 0.05.