Effects of plant essential oil supplementation on growth performance, immune function and antioxidant activities in weaned pigs

Background The aim of this study was to determine the effects of plant essential oil supplementation on growth performance, immune function and antioxidant activities in weaned pigs. Methods In the study, 24 weaned pigs were used to explore the effects of plant essential oil (PEO) on growth performance, immune properties and antioxidant activities. Pigs were fed with a basal diet (CON) or basal diet containing different concentrations of PEO (PEO50: 50 ppm; PEO100: 100 ppm; PEO200: 200 ppm). After 3 weeks, all pigs were slaughtered and blood and tissue samples were collected for biochemical analysis. Results The results showed that PEO supplementation quadratically increased body weight gain (BWG) (P = 0.031), linearly (P <  0.05) and quadratically (P <  0.05) decreased F:G. In addition, IgG increased linearly (P <  0.05) and IgM increased linearly (P <  0.05) and quadratically (P < 0.05) as PEO supplementation. Similarly, MDA in serum, jejunal mucosa and pancreas were linearly decreased (P < 0.05) and GSH in serum (linear and quadratic, P < 0.05), duodenal mucosa (linear and quadratic, P < 0.05) and in ileal mucosa (linear and quadratic, P < 0.05) were notably increased. Futhermore, antioxidant-related genes expression levels of GST in spleen (linear and quadratic, P < 0.05), GPX1 (quadratic, P < 0.05) and SOD1 (linear, P < 0.05) in spleen and GST in liver (quadratic, P < 0.05) were markedly upregulated by PEO supplementation increasing. Conclusions These results suggest that PEO improves growth performance, immune function, and antioxidant activities in weaned pigs, and it may also relieve weaning stress if used as a feed additive in the livestock industry. And that supplementation 200 ppm PEO in diet would seem to be economically feasible.


Background
Recent studies have indicated that weaning can induce oxidative stress in pigs, resulting in oxidative damage [1]. Weaning stress has been reported to disrupt intestinal health, cause diarrhea, and to reduce growth and immunity in pigs [2][3][4]. Over the past number of decades, antibiotics, zinc oxide, and copper sulfate have been widely utilized in the swine industries for their effects in reducing diarrhea and improving immunity in weanling pigs [5][6][7]. However, the abuse of these additives has led to antibiotic resistance and heavy metal residues in livestock products.
The rising incidence of these serious problems has compelled research institutes and farmers alike to search for safe feed additives [8][9][10].
Many essential oils from plants, either extracted or in their natural form, are used for their antioxidative properties, which are mainly due to phenolic compounds in the oil or in other phytochemical fractions [11,12]. Some nonphenolic substances also exhibit considerable antimicrobial and antioxidative potential [13,14]. Previous studies have reported that essential oils may improve nutrient digestibility, as well as intestinal morphology and microflora [15][16][17]. Other studies have reported that essential oil supplementation improved the nutritional value and oxidative stability of fat, meat, and eggs, resulting in longer shelf-life [18][19][20][21][22][23]. These previous findings regarding animal products led us to hypothesize whether essential oils could improve systemic redox balance and reduce oxidative injury induced by weaning stress in young pigs.
Cinnamaldehyde is an organic compound with the formula C 6 H 5 CH=CHCHO, and thymol is a natural monoterpene phenol with the formula C 10 H 14 O (Fig. 1). Although several studies have indicated that cinnamaldehyde and thymol can improve growth performance, nutrient digestibility, and intestinal morphology, and stabilize the microflora of weaned pigs and poultry [11,17,24,25], the effects of cinnamaldehyde and thymol on antioxidant activity and immune function in weaned pigs is still unclear. Therefore, the purpose of this study was to explore the effects of dietary supplementation with plant essential oil on both immune function and antioxidant activity in weaned pigs.

Diets and feeding management
Diets were corn-soybean based diets and formulated according to National Research Council 2012 requirements [26]. Ingredients and nutrient composition of experimental diets are shown in Table 1. Diets were fed in mash form throughout the experiment.
The experiment was carried out at the Research Base of the Institute of Animal Nutrition of Sichuan Agricultural University. All the pigs were housed in an environmentally controlled nursery room in individual metabolism cages (1.5 m × 0.7 m × 1.0 m). The pigs had free access to diet and water throughout the 2-week feeding trial, and were fed with the experimental diets 4 times daily at 08:00, 12:00, 16:00 and 20:00. Temperature was gradually reduced from 28°C to 23°C, and the humidity was controlled between 50 and 60%. Pigs were weighed at 08:00 on an empty stomach on days 0 and 14.

Sample collection
Blood samples were collected from the portal vein precava into Vacuum pick blood vessels without anticoagulation (Axygen Biotechnology Co. Ltd) at 8:00 in the morning on day 14. From each sample, serum were collected by centrifuging the blood (3500 g, 4°C, 10 min) and immediately stored at − 20°C. All pigs were slaughtered by exsanguination according to protocols approved by the Sichuan Agricultural University Animal Care Advisory Committee. Mucosa of duodenum, jejunum and ileum, spleen, liver and pancreas were collected and snap frozen in liquid N 2 and then stored at − 80°C for assay.

Growth performance
Individual initial and final body weight were recorded on day 0 and day 14. Body weight gain (BWG) was calculated as final body weight subtract initial body weight. Average daily gain (ADG) was calculated as weight (kg)/ number of days from initial to final weight.

Immunity parameters
The levels of total protein (TP), albumin (ALB), IgG and IgM in serum were examined using the Hitachi 7020 Automatic Analyzer (Tokyo, Japan). The level of IgA in serum was measured using an ELISA test kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer's instructions, the absorbance was determined with a 96-well microtiter plate reader Spectramax M2 (Molecular Devices, Sunnyvale, CA).

Total protein concentration analysis
Total protein assay kit BCA (A045-3) was purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Apple green BCA reacted with protein at 37°C for 30 min and formed a purple compound featuring absorbance at 562 nm. TP results are expressed as aprot per litre (gprot/L) of tissues.

GSH content analysis
GSH content was measured according to previous report. [4] Reduced GSH assay kit (A006-2, Nanjing Jiancheng Bioengineering Institute (Nanjing, China)) was used according manufacturer's instructions. GSH content was expressed in gGSH/L and mgGSH/gprot in serum and tissues respectively by using commercial GSH as a standard.

CAT activity analysis
CAT activity was measured according to previous report [27]. CAT assay kit (A007-1, Nanjing Jiancheng Bioengineering Institute (Nanjing, China)) was used according manufacturer's instructions. The reaction of CAT decomposing H 2 O 2 was rapidly stopped reaction after adding ammonium molybdate. The rest of H 2 O 2 reacted with ammonium molybdate and formed a pale yellow complex compound featuring absorbance at 405 nm. CAT results were expressed in U/mL and U/ gprot in serum and tissues respectively.

T-AOC activity analysis
T-AOC activity was measured according to previous report [28]. Total antioxidant capacity assay kit (A015-1, Nanjing Jiancheng Bioengineering Institute (Nanjing, China)) was used according manufacturer's instructions. T-AOC activity was expressed in U/mL and U/gprot in serum and tissues respectively.

MDA content analysis
MDA content was analyzed as described by previous report [29]. MDA assay kit (TBA method) (A003-1, Nanjing Jiancheng Bioengineering Institute (Nanjing, China)) was used according manufacturer's instructions. MDA reacted with thiobarbituric acid (TBA) formed a red-complex compound featuring absorbance at 532 nm. MDA results were expressed in nmol/mL in serum and tissues.

RNA isolation and real-time quantitative PCR
Total RNA from samples of spleen, liver and pancreas was extracted using TRIzol reagent (TaKaRa, Dalian, China) according to the manufacturer's instructions. RNA concentration was measured by Nanodrop 2000 (Thermo Fisher Scientific, Wilmington,DE, USA). The integrity of RNA was verified by eletrophoretic analysis. Complementary DNA (cDNA) was achieved by reverse transcription with 2 μg RNA sample using the PrimeScript™ RT reagent Kit (TaKaRa, Dalian, China) according to the manufacturer's instructions. 20 μL final reaction volume of cDNA was then diluted to 250 μL using nuclease-free water and stored at − 20°C. The cDNA was used as the template for PCR. Real-time quantitative PCR was performed on cDNA using the ABI PRISM 7500 Fast Sequence Detection System for ninety-sixwell plates (Applied Biosystems). The primers of β-actin, superoxide dismutase 1 (SOD1), CAT, glutathione peroxidase 1 (GPX1), glutathione transferase (GST), glutathione reductase (GR), nuclear factor E2-related factor 2 (Nrf2), Kelch-like ECH-associated protein 1 (Keap-1) are shown in Table 2. The gene β-actin was used as an internal control. All the genes of each sample were repeated in triplicate. For each reaction, 5 μL of freshly SYBR® Premix Ex Taq™ II (Tli RNaseH Plus, 2×), 1 μL forward primers (4 mmol/L) and 1 μL reverse primers (4 mmol/L), 1 μL of cDNA and 2 μL nuclease-free water were added and made up to final volume of 10 μL. The PCR programme was as follow: a predegeneration at 95°C for 10 min for one cycle, followed by denaturation at 95°C for 5 s, annealing temperature at 60°C for 45 s, extension temperature at 72°C for 10 s for forty cycles. After amplification, the melting peaks of the amplification products were determined by melting curve which indicated only one expected amplification products had been generated. Melting curve conditions were as follows: 1 cycle of denaturation at 95°C for 10 s and then 65°C changed to 95°C with a temperature change velocity of 0.5°C/s. The standard curve of each gene was run in triplicate for obtaining reliable amplification efficiency values. The correlation coefficients of all the standard curves were > 0.99, and the amplification efficiency values were between 90 and 110%. Each primer pair used yielded a single peak in the melting curve and a single band with the expected size in agarose gel. The mean Ct values of duplicates of each sample were used for calculations. The relative gene expressions compared with the housekeeping gene b-actin were calculated by 2 -CT [31].

Statistical analysis
All data were analysed using SPSS 20.0 software (SPSS, Inc.) by curve estimation model of regression procedure.
The effect of PEO supplementation was determined by linear and quadratic effects in individual pig unit. All data were expressed as mean ± standard error. The significance level for all analyses was set at P < 0.05, with a trend of 0.05 ≤ P ≤ 0.01.

Results
Effect of PEO on growth performance As indicated in Table 3, the BWG increased quadratically (P = 0.031), PEO200 increased the BWG 0.52 kg than CON. In addition, F:G also decreased linearly (P = 0.008) and quadratically (P = 0.007) as supplementation of PEO increased. PEO200 decreased 0.13 than CON in F: G.

Effect of PEO on blood biochemical parameters and serum immunoglobulins
The effect of PEO supplementation on the level of serum TP, ALB, IgG, IgA and IgM were presented in Table 4. IgG increased linearly (P = 0.029) and IgM increased linearly (P = 0.003) and quadratically (P = 0.007) as supplementation of PEO increased. And then, supplementation of PEO tended quadratically (P = 0.098) increase IgM and linearly (P = 0.098) and quadratically (P = 0.070) increase ALB. Moreover, serum cholesterol was linearly (P = 0.039) as PEO supplementation.

Effect of PEO on serum antioxidant activity
There was a significant increase in serum GSH with increasing levels of PEO (linear, P < 0.01; quadratic,

Effect of PEO on intestinal mucosal antioxidant activity
The effect of PEO supplementation on the level of intestinal mucosa TP, GSH, T-AOC, T-SOD, MDA and CAT were presented in Table 6. There was a significant increase in GSH with increasing levels of PEO in duodenal mucosa (linear, P = 0.007; quadratic, P = 0.029) and in ileal mucosa (linear, P = 0.011; quadratic, P = 0.032). MDA in jejunal mucosa decreased linearly (P = 0.042) as supplementation of PEO increased.

Effect of PEO on splenic, hepatic and pancreatic antioxidant activity
The levels of GSH, CAT, T-AOC, T-SOD and MDA in spleen, liver and pancreas were measured (Table 7). GSH in pancreas increased linearly (P = 0.007) and quadratically (P = 0.032) as supplementation of PEO increased. There was a decrease at 100 ppm (linear, P = 0.032) in MDA of pancreas with no further decrease at 200 ppm.

Effect of PEO on the expression of critical antioxidantrelated genes
Gene expression of GR, GST, GPX1, CAT, SOD1, Nrf1 and Keap1 was measured ( Table 8). Expression of GST in spleen increased linearly (P = 0.012) and quadratically (P = 0.007) as supplementation of PEO increased. There was a tendency to increase expression level of Keap1 with increasing levels of PEO in spleen (quadratic, P = 0.085). An increased in GPX1 (quadratic, P = 0.029), CAT (quadratic, P = 0.051) and SOD1 (linear, P = 0.029) was observed in the spleen of pigs supplemented with 200 ppm PEO. Moreover, the expression of GST in liver increased quadratically (P = 0.049) as supplementation of PEO increased. However, the expression of Nrf2 in liver decreased linearly (P = 0.023) and quadratically (P = 0.029) as supplementation of PEO increased. Furthermore, there was no effect on the expression of GR, GST, GPX1, CAT, SOD1, Nrf1 and Keap1 in pancreas with increasing dietary inclusion level of PEO.

Discussion
Weaning stress syndrome was an inevitable problem, which could lead weaning stress, oxidative stress, and  adversely affects intestinal health, leads to diarrhea, and reduces growth and immunity in pigs [2][3][4]. Oxidative stress is caused by excess oxidative radicals, including reactive oxygen species, which damage DNA, bio-membrane lipids, and proteins, and also impair tissue function [32,33]. This may be the reason that weaning stress syndrome reduces growth performance and economic benefit in pig production. Thymol and cinnamaldehyde are concentrated hydrophobic liquids containing volatile aromatic compounds extracted from plants, which have unique chemical structures (Fig. 1) and could be used as natural antioxidants [11,34]. In this study, dietary supplementation of PEO at a dose of 200 ppm quadratically improved BWG of pigs after weaning. As well as, ADG tended to increase quadratically as 200 ppm PEO supplementation increased. Because of there was no effect on average daily feed intake (ADFI) with the increasing PEO addition. So the most important reason for growth performance improvement was feed efficiency. F:G decreased linearly   [17,24]. The improvement in nutrient absorption may be partly explained by increased secretions of saliva, bile and enhanced enzyme activity [35][36][37][38]. Moreover, PEO supplementation has been reported to improve the immune status of pigs after weaning, as indicated by increased serum immunoglobulin levels [15,17]. Our results indicated that PEO supplementation increased the levels of ALB, IgA, and IgG in serum, and 200 ppm PEO addition got the highest levels. It is well known that IgG offers newborn pigs extended systemic protection, while IgA and IgM offer transient luminal protection [39]. During the first few days after weaning, piglets often experience low feed intake or starvation issues that can represent a major challenge for the producer. Therefore, the administration of 200 mg/kg PEO to piglets during this time probably improve their immune status and prevent oxidative stress.
Oxidative stress is caused by excess oxidative radicals, including reactive oxygen species (ROS) and reactive nitrogen species (RNS), which damage DNA, bio-membrane lipids, proteins, and other macromolecules [32]. However, excess oxidative radicals can be eliminated by antioxidants, including nonenzymatic components and a series of enzymes. Within the enzymatic antioxidant system, superoxide dismutase and glutathione peroxidase are the most important compounds [21], working together to detoxify superoxide anions and hydrogen peroxide in cells [33]. Superoxide dismutase catalyzes superoxide anions to produce hydrogen peroxide and molecular oxygen. Glutathione peroxidase normally converts H 2 O 2 to water [21]. The capabilities of the non-enzymatic antioxidant defense system are often measured as the total antioxidant capacity [40]. Furthermore, the level of malondialdehyde, a major product of lipid peroxidation, is an effective marker of oxidative stress in sepsis [21].
In the present study, we measured GSH, CAT, MDA, and T-SOD levels, as well as T-AOC, in serum, intestinal mucosa, spleen, liver, and pancreas. Present results indicated that PEO supplementation significantly increased GSH levels in serum, duodenal and ileal mucosa and pancreas, T-SOD activity in jejunal mucosa and spleen, and significantly decreased MDA levels in serum, jejunal mucosa and pancreas. The results of this study were consistent with previous reports that T-AOC was improved and plasma MDA level was decreased in pigs receiving supplemental PEO [15,41,42]. Previous study have confirmed that PEO could save the depletion of GSH glutathione, CAT catalase, T-AOC total antioxidant capacity, MDA methane dicarboxylic SOD and GSH-Px or enhance their capabilities [21]. The increased GSH and decreased MDA levels in serum indicated that whole-body antioxidant status was improved, and lipid peroxidation was reduced. In addition, the reduced MDA levels and improvements in GSH levels and T-SOD activity suggested that PEO could enhance both the non-enzymatic and enzymatic reactions of the antioxidant defense system. Various natural antioxidant extracts have been used to protect pigs from weaning stress in intensive pig production [1]. Our results suggests that dietary PEO supplementation may reduce oxidative stress and have the potential to ameliorate the adverse effects of early weaning syndrome. In order to elucidate the mechanism of PEO antioxidant activity in weaned pigs, we measured the expression of SOD1, CAT, GPX1, GST, GR, Nrf2, and Keap-1 in the spleen, liver, and pancreas. Keap-1 is the cytosolic protein with which the transcription factor Nrf2 is associated, and which functions to protect against oxidative stress. Oxidative stress modifies Keap-1 at redox-sensitive SH groups, leading to the liberation and nuclear translocation of Nrf2. Subsequently, Nrf2 binds to the ARE promoter sequence of antioxidant enzymes. In this way, Nrf2 coordinates cytoplasmic responses to oxidative stress [43]. A previous study reported that essential oils increased the mRNA expression of jejunal GPx1 and SOD1, ileal GST, and colonic GPX1, SOD1, and Keap-1 [44]. Similarly, the present results indicated that PEO supplementation upregulated the expression of GST, GPX, CAT, and SOD1 in the spleen and the expression of GST and Nrf2 in the liver. It can be speculated that the different terpene compounds in PEO can modify Keap 1 at sensor -SH groups through chemical reactions [45]. Oxidative stress modifies Keap1 at redox-sensitive -SH groups, which leads to Nrf2 liberation and its nuclear translocation. Subsequently, Nrf2 Values are means ± S.E, n = 6 b SOD1, superoxide dismutase 1; CAT, catalase; GPX1,glutathione peroxidase 1; GST, glutathione transferase; GR, glutathione reductase; Nrf2, nuclear factor E2-related factor 2; Keap-1, Kelch-like ECH-associated protein 1 binds to the ARE promoter sequence of antioxidant enzymes. In this way, Nrf2 coordinates cytoplasmic responses to oxidative stress [43]. The changes in the expression of genes associated with oxidation and antioxidant compounds may explain the findings in serum and tissue samples. The spleen is a vital part of the immune system and the liver is important in detoxification and metabolism in pigs [46,47]. The improved splenic and hepatic antioxidant capacities may be reflected in the immunity and health of weaned pigs. Although the PEO used in our study has been confirmed to improve the antioxidant capacities of serum and other tissues, the molecular mechanisms through which PEO impacts the upstream Nrf2 pathways and then modulates antioxidant capacity remains to be further investigated.

Conclusions
In conclusion, our results suggest that dietary PEO supplementation improved growth performance, immune function, and antioxidant status in weaned pigs. The benefits observed may be mediated by upregulation of antioxidant-related genes in the spleen and liver. And that supplementation of the PEO preparation at the levels of 200 mg/kg diet would seem to be economically feasible. This study not only provides new insights into the role of PEO in improving growth performance, immune function, and antioxidant capacity in weaned pigs, but also reveals a potential candidate to replace the antibiotics conventionally used in the livestock industry.

Availability of data and materials
The dataset supporting the conclusions of this article is included within the article.
Authors' contributions JH, DC, GC and YL were involved in the study design; XZ participated in data analysis, interpretation, XZ and YW performed the animal management; GS drafted the manuscript. All authors read and approved the final manuscript.

Ethics approval
The animal experiment followed the actual law of animal protection and was approved by the Animal Care and Use Committee of the Sichuan Agricultural University and was performed in accordance with the National Research Council's Guide for the Care and Use of Laboratory Animals.