Dietary mixed plant oils supplementation improves performance, serum antioxidant status, immunoglobulin and intestinal morphology in weanling piglets

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Dietary mixed plant oils supplementation improves performance, serum antioxidant status, immunoglobulin and intestinal morphology in weanling piglets S.F. Long, T.F. He, Li Liu, X.S. Piao* State Key Laboratory of Animal Nutrition, College of Animal Science and Technology, China Agricultural University, Beijing 100193, China

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ABSTRACT

Keywords: Antioxidant status Digestibility Immunity Mixed plant oils Performance Piglets

The aim of this study was to evaluate the effect of two novel mixed plant oils on performance, serum immunity, antioxidant capacity and intestinal morphology in weanling piglets compared with soybean oil (SO). A total of 108 piglets [Duroc × (Landrace × Yorkshire), weaned at d 28, weighting 8.80 ± 1.02 kg] were randomly allotted into 1 of 3 dietary treatments with 6 replicate pens per treatment, 3 barrows and 3 gilts per pen. This experiment contained phase 1 (d 0–14) and 2 (d 14–28). Dietary treatments included a control diet (CON; corn-soybean meal basal diet + 5% SO in phase 1 or 4% SO in phase 2), mixed plant oil 1 diet (MPO1; basal diet + 5% MPO1 in phase 1 or 4% MPO1 in phase 2; a mixture of 10% coconut oil, 15% corn oil, 15% linseed oil, 15% peanut oil, 20% palm oil, and 25% SO), and mixed plant oil 2 diet (MPO2; basal diet + 5% MPO2 in phase 1 or 4% MPO2 in phase 2; a mixture of half MPO1 and half extruded corn). Compared with CON, piglets fed MPO (MPO1 or MPO2) had increased (P < 0.05) average daily gain and feed efficiency in phase 1 and overall (d 0–28), and improved (P < 0.05) serum superoxide dismutase (SOD) content on d 14. These piglets also had higher (P < 0.05) serum IgG, SOD, glutathione peroxidase contents, villus height in duodenum and jejunum, and apparent total tract digestibility (ATTD) of ether extract on d 28. Piglets fed MPO2 showed a higher (P < 0.05) IgM content on d 14 and growth hormone content in serum on d 28 compared with CON. The results indicate that mixed plant oils can be better energy feed than soybean oil in improving growth performance, serum immunity, antioxidant capacity, apparent total tract digestibility of ether extract and intestinal morphology in weanling piglets.

1. Introduction Weanling piglets are easily prone to weaning stress due to the sudden changes of environment, nutrition and psychology, which can cause a decrease in digestive enzyme activity, feed intake and growth performance (Ferrara et al., 2017). The rapidly decreased activities of pancreatic lipase and pancreatic digestive enzymes may also reduce digestion and absorption of energy feeds, such as animal fats and plant oils (Gu and Li, 2003). Therefore, adding lipids (including animal fats and plant oils) to swine diets can not only help supply concentrated energy, highly digestive essential fatty acids and fat soluble vitamins, but also increase the palatability of Abbreviations: ADFI, average daily feed intake; ADG, average daily gain; ATTD, apparent total tract digestibility; CP, crude protein; DM, dry matter; EE, ether extract; FE, feed efficiency; GE, gross energy; Lys, lysine; MCFA, medium chain fatty acid; MUFA, monounsaturated fatty acids; Met, methionine; MPO1, mixed plant oil 1; MPO2, mixed plant oil 2; PUFA, polyunsaturated fatty acids; Thr, threonine; Trp, tryptophan ⁎ Corresponding author. E-mail address: [email protected] (X.S. Piao). https://doi.org/10.1016/j.anifeedsci.2019.114337 Received 4 January 2019; Received in revised form 4 October 2019; Accepted 25 October 2019 0377-8401/ © 2019 Elsevier B.V. All rights reserved.

Please cite this article as: S.F. Long, et al., Animal Feed Science and Technology, https://doi.org/10.1016/j.anifeedsci.2019.114337

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swine diet and therefore improve the feed efficiency and intestinal health for weanling piglets (Pettigrew et al., 1991; Rossi et al., 2010). Moreover, monounsaturated fatty acid (MUFA) and polyunsaturated fatty acid (PUFA) in animal fats or plant oils also had nutritional and physiological effects and could help alleviate weaning stress in early weaning piglets (Zentek et al., 2011). Dietary fat supplementation can supply piglets with more energy, essential fatty acids and fat-soluble vitamins than carbohydrate (Lerma-Reyes et al., 2018). Animal fats (e.g. poultry oil, fish oil and lard) are easily to be oxidant because they contain plenty of unsaturated fatty acid, whereas plant oils (e.g. palm, coconut, linseed, corn, soybean, peanut oils) are environmentally and economically sustainable as they have high contents of saturated fatty acid (SFA), MUFA, and a little PUFA (Teoh and Ng, 2016). Therefore, plant oils are widely used as important sources of energy feeds in weanling piglets. Corn oil, sunflower oil and SO are rich in n-6 fatty acids, mainly as linoleic acid, whereas linseed oil is rich in α-linolenic acid. These plant oils can be absorbed and metabolized efficiently, and have physiological benefits beyond their energetic value. Plant oils can allow the rapid provision of energy for enterocytes and intermediary hepatic metabolism, which devotes to the improvement of growth performance in weanling piglet (Zentek et al., 2011, 2012). Previous studies also show dietary linseed oil supplementation can increase immunity, modify fatty acid profile, and improve growth performance in piglets compared with soybean oil (SO) (Chen et al., 2016). Moreover, adding 3% mixed plant oils (a mixture of linseed oil, palm oil, SO, coconut oil and corn oil) replacing 3% SO in broiler diet can effectively improve performance, serum composition and fatty acid deposition (Long et al., 2018a). However, a study carried by Vilarrasa et al. (2015) shows dietary supplementation with 10% palm oil and SO has no significant improvement of nutrients digestibility or performance in weanling piglets. These different results of mixed plant oils may be related to the effectiveness of plant oils differences, the type of fatty acid intake in basal diet, the method of determination, and the difference of fatty acid composition (Li et al., 1990). Moreover, oils used in animal production not only vary in fatty acid composition because of their origin but also contain various concentrations of primary and secondary lipid peroxidation products depending on fatty acid composition, storage length and conditions, and effects of processing (Canakci, 2007). Although there are many studies showed different combination of fatty acid composition in plant oils, such as SFA and unsaturated fatty acids, can affect the nutrient digestibility and performance for post-weaning piglets (Wiseman et al., 1990), few studies focus on finding novel effective combination of six different types of high-quality plant oils, and their effects on performance, nutrients digestibility, and intestinal health in piglets after weaning. In this study, the hypothesis is that supplementation of a combination of different plant oils in corn-soybean meal diet can increase performance and nutrient digestibility of weaned piglets via the improvement of intestinal development and immunity. Therefore, the aim of the present study is to evaluate the effect of two novel mixed plant oils on performance, nutrient digestibility, serum immunoglobulin, antioxidant capacity and intestinal morphology of weanling piglets in comparison with SO. 2. Material and methods All the procedures used in this experiment were approved by the Institutional Animal Care and Use Committee of China Agricultural University (Beijing, China), and executed in the National Feed Engineering Technology Research Center of the Ministry of Agriculture Feed Industry Center Animal Testing Base (Hebei, China). 2.1. Mixed plant oils The two novel mixed plant oils used in this research were produced in Shandong Zhongda Agricultural Science and Technology Co., Ltd. (Binzhou, Shandong, China). The MPO1 is composed of 10% coconut oil, 15% corn oil, 15% linseed oil, 15% peanut oil, 20% palm oil, as well as 25% SO. The MPO2 is composed of half MPO1 and half extruded corn. The fatty acid composition of MPO is shown in Table 1. The MUFA of MPO1 and MPO2 are 17.52% and 10.22%, PUFA of MPO1 and MPO2 are 38.84% and 23.21%, while n-6 PUFA are 36.34% and 21.72%, respectively. 2.2. Experimental animals and design A total of 108 piglets [Duroc × (Landrace × Yorkshire), weighting at 8.80 ± 1.02 kg] weaned at 28 d of age were allotted into 3 treatments in a randomized complete block design with 6 replicate pens per treatment, and 6 piglets (3 barrows and 3 gilts) per pen. The experiment contained phase 1 (d 0–14) and 2 (d 14–28). Dietary treatments included a control diet (CON; corn-soybean meal basal diet + 5% SO in phase 1 or 4% SO in phase 2), mixed plant oil 1 diet (MPO1; basal diet + 5% MPO1 in phase 1 or 4% MPO1 in phase 2), and mixed plant oil 2 diet (MPO2; basal diet + 5% MPO2 in phase 1 or 4% MPO2 in phase 2). As shown in Table 2, nutrients in the diet met the recommended requirements of NRC (2012), and we modulated the corn and soybean meal levels to keep the digestible energy (DE) and crude protein (CP) levels remained similar in all diets of phase 1 and 2. All the piglets were raised in pens (1.2 m × 2 m) fitted with a duckbill drinker, an adjustable stainless steel feeder and plastic slatted floors. Inside the pen, these piglets also had free access to feed (in mash form) and water ad libitum. The environment in the pig house, including the contents of CO2 and ammonium in the air, ventilation intensity, humidity and temperature, was controlled automatically. The average temperature in the room was controlled at 24–26 °C, and the average relative humidity was maintained at 60–70%. On d 0, 14 and 28, all the piglets and feed were weighed to calculate the performance, including the average daily gain (ADG), average daily feed intake (ADFI) as well as feed efficiency (FE). From d 0–28, the diarrhea score in each pen was recorded twice every day according to a scoring system following by Long et al. (2018b). The determination of diarrhea rate was mainly depended on the average diarrhea score, following the formula: Diarrhea rate (%) = diarrhea days × the number of diarrhea pigs/ (experiment days × the total number of pigs). 2

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Table 1 Chemical composition of mixed plant oils (%, as-fed basis). Item

MPO11

MPO21

Day matter Ether extract Fatty acids C12:0 C14:0 C16:0 C18:0 C18:1n9 C18:2n6 C18:3n3 C20:0 SFA2 MUFA2 PUFA2 n-6 PUFA2 n-3 PUFA2 SFA / PUFA2 n-6 / n-3 PUFA2

0.36 98.95

4.59 51.53

1.77 1.02 17.40 2.80 17.52 36.34 2.50 0.24 23.72 17.52 38.84 36.34 2.50 0.61 14.54

0.80 0.53 9.79 1.62 10.22 21.72 1.49 0.14 12.08 10.22 23.21 21.72 1.49 0.52 14.58

Note: 1 MPO1: mixed plant oil 1; MPO2: mixed plant oil 2. 2 These values are analyzed. Saturated fatty acid (SFA) = C14: 0 + C16: 0 + C17: 0 + C18: 0 + C20: 0; Monounsaturated fatty acid (MUFA) = C16: 1n-7 + C18: 1n-9; Polyunsaturated fatty acid (PUFA) = C18: 2n-6 + C18: 3n-6 + C20: 4n-6 + C18: 3n-3 + C20: 5n-3 + C22: 6n-3; N-6 PUFA = C18: 2n-6 + C18: 3n-6 + C20: 4n-6; N-3 PUFA = C18: 3n-3 + C20: 5n-3 + C22: 6n-3.

2.3. Chemical analysis During this experiment, a total of 2 kg representative feed samples were collected weekly. The fatty acids compositions of diets were tested according to the procedure of Long et al. (2018a). From d 26–28, the rectal palpation was used to make sure approximately 400 g of fresh feces were collected using the grab sample technique. All the fecal samples were frozen at −20 °C immediately after collection until analysis. The feces collected during 3 days was pooled by pen and dried at 65 °C for 72 h. Before analysis, all these dried feces and feed samples were ground to pass through a 1-mm sieve. The day matter (DM) and ether extract (EE) in MPO, as well as the CP, DM and EE in feed and feces were measured using the methods of AOAC (2007). The fatty acid compositions of MPO were measured according to the procedure of Long et al. (2018a). The gross energy (GE) in feed and feces was determined by an automatic isoperibolic oxygen bomb calorimeter (Parr 1281, Automatic Energy Analyzer; Moline, IL). Moreover, an atomic absorption spectrophotometer (Z-5000; Hitachi, Tokyo, Japan) was used to determine the content of chromium in feed and feces. The calculation of nutrient digestibility was as follows: Apparent total tract digestibility (ATTD)nutrient = 1- (Crdiet × Nutrientfeces) / (Crfeces× Nutrientdiet). A total of approximate 8 mL blood sample of piglet near the average group body weight in each pen was collected via jugular vein puncture into vacutainer at 7:00 am on d 14 and d 28 (Becton Dickinson Vacutainer Systems, Franklin Lakes, NJ). After stewing for 3 h, all the collected blood samples were centrifuged at 3000 x g for 10 min at 4 °C to get the serum samples, which were also stored at −20 °C until analysis. An ELISA kit (IgG, IgM and IgA quantitation kit; Bethyl Laboratories, Inc., Texas) was used to determine the concentrations of serum immunoglobulins (including immunoglobulin G, immunoglobulin M, and immunoglobulin A). Moreover, the contents of serum superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT), malondialdehyde (MDA), urea, total cholesterol (TC), total glyceride(TG), total protein (TP), glucose (GLU), insulin (INS), gortisol (COR) and growth hormone (GH) were determined by spectrophotometric methods using a spectrophotometer (LengGuang SFZ1606017568, Shanghai, China) following the instructions of the kit’s manufacturer (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). On d 28, the aseptic duodenal, jejunal and ileal samples (about 5 cm fragment in the middle of each selected intestine, selected duodenum as the proximal 1/3 of the small intestine, jejunum as the 1/3 mid and ileum as 1/3 distal part) were collected from slaughtered barrows (near the average group body weight) selected in each pen for the determination of intestinal morphology. According to the procedure of Xu et al. (2018), 10% neutral buffered formalin was used to fix rapidly these histological samples for slicing. After 48 h of fixation, the sections of intestinal tissues were washed, excised, dehydrated, as well as embedded in the paraffin wax, and then 5 transverse sections were slicing, installed on glass slides and dyed with eosin and hematoxylin. At least 20 orientated villi and their adjoining crypts were selected randomly on each slice and measured to calculate the average villus height and crypt depth via a light microscope in small caps using a calibrated 10-fold eyepiece graticule. The ratio of villus height to crypt depth was calculated and used for further analysis.

3

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Table 2 Composition and nutrient levels of basal diets (%, as-fed basis). Ingredients

Corn Soybean meal, 43% Extruded soybean Fish meal Spray dried plasma protein Soybean oil Mixed plant oil 1 Mixed plant oil 2 Dicalcium phosphate Limestone Salt L-lysine HCl, 78% DL-Methionine, 98% L-Threonine, 98% L-Tryptophan, 98% Chromic oxide Vitamin-mineral premix2 Nutrient composition3 Digestible energy, MJ/kg Gross energy, MJ/kg Crude protein Calcium Digestible phosphorous Dry matter Lysine Methionine Threonine Tryptophan

Phase 1 (d 0 – 14)

Phase 2 (d 14 – 28)

CON1

MPO11

MPO21

CON

MPO1

MPO2

64.36 18.00 1.50 4.00 3.50 5.00 – – 0.90 1.02 0.30 0.44 0.10 0.11 0.02 0.25 0.50

62.87 14.00 7.00 4.00 3.50 – 5.00 – 0.92 1.00 0.30 0.43 0.10 0.11 0.02 0.25 0.50

59.50 6.50 18.00 4.00 3.50 – – 5.00 0.88 1.00 0.30 0.38 0.09 0.09 0.01 0.25 0.50

67.66 20.00 0.00 3.00 2.00 4.00 – – 0.61 1.02 0.30 0.43 0.09 0.12 0.02 0.25 0.50

66.21 17.50 4.00 3.00 2.00 – 4.00 – 0.60 1.02 0.30 0.41 0.08 0.11 0.02 0.25 0.50

63.48 11.50 12.80 3.00 2.00 – – 4.00 0.60 1.00 0.30 0.37 0.08 0.10 0.02 0.25 0.50

14.82 17.58 21.52 0.80 0.80 89.55 1.37 0.41 0.94 0.23

14.82 17.76 21.41 0.80 0.40 89.64 1.61 0.41 0.87 0.23

14.80 17.71 21.40 0.80 0.40 89.57 1.33 0.38 0.94 0.22

14.61 17.41 19.30 0.70 0.33 89.27 1.08 0.37 0.85 0.21

14.60 17.46 19.34 0.70 0.33 89.22 1.37 0.36 0.88 0.21

14.58 17.32 19.52 0.70 0.33 89.72 1.17 0.41 0.85 0.22

Note: 1 CON: control; MPO1: mixed plant oil 1; MPO2: mixed plant oil 2. 2 Premix provided the following per kg of feed: vitamin A, 12,000 IU; vitamin D3, 2500 IU; vitamin E, 30 IU; vitamin K3, 30 mg; vitamin B12, 12 μg; riboflavin, 4 mg; pantothenic acid, 15 mg; niacin, 40 mg; choline chloride, 400 mg; folic acid, 0.7 mg; vitamin B1, 1.5 mg; vitamin B6, 3 mg; biotin, 0.1 mg; Mn, 40 mg; Fe, 90 mg; Zn, 100 mg; Cu, 8.8 mg; I, 0.35 mg; Se, 0.3 mg. 3 Gross energy, crude protein, dry matter, lysine, methionine, threonine and tryptophan were analyzed values, the rest were calculated values.

2.4. Statistical analysis The MIXED model of SAS (version 9.2, 2008) was used for variance analysis of all the data. The dietary treatments were fixed effects, while sex and body weight of pigs were the random effects. For the analysis of growth performance, fecal score and diarrhea rate, the pen was treated as the statistical unit, whereas for the analysis of other data, individual pig was taken as the statistical unit. The Student-Neuman-Keul’s Multiple Range Tests was used for determining the statistical differences among all the treatments. Significant difference between the mean value was defined at P ≤ 0.05, while a trend for the significance between the mean value was designated at 0.05 < P ≤ 0.10. 3. Results 3.1. Fatty acids composition in experimental diets The fatty acids composition including SFA, MUFA, PUFA and their ratios in experimental diets was tested and showed in Table 3. In phase 1, the n-6 / n-3 PUFA ratios of CON, MPO1 and MPO2 diets were about 15, 12 and 6.4, respectively, and these ratios were about 18, 15 and 8 in phase 2, respectively. 3.2. Performance and diarrhea rate According to Table 4, piglets fed MPO (MPO1 or MPO2) had increased (P < 0.05) ADG and FE in phase 1 and overall (d 0–28), while there was no significant difference of ADFI in piglets fed MPO in comparison with CON. Although there is no significant difference of fecal score and diarrhea rate among all the treatments, the diarrhea rate of piglets fed MPO showed a reduction of 70%, 44% and 63% respectively in phase 1, 2 and overall compared with CON. 4

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Table 3 The fatty acid composition of the experimental diets (g / 100 g fatty acid). Item

C12:0 C14:0 C16:0 C18:0 C18:1n9 C18:2n6 C18:3n3 C20:0 SFA2 MUFA2 PUFA2 n-6 PUFA2 n-3 PUFA2 SFA / PUFA2 n-6 / n-3 PUFA2

Phase 1 (d 0 – 14)

Phase 2 (d 14 – 28)

CON1

MPO11

MPO21

CON1

MPO11

MPO21

0.00 0.23 5.87 1.06 8.71 17.11 1.14 0.07 7.23 8.71 18.25 17.11 1.14 0.40 14.98

0.09 0.28 6.61 1.19 9.37 15.56 1.29 0.09 8.27 9.37 16.85 15.56 1.29 0.49 12.04

0.04 0.27 7.06 1.53 10.56 13.41 2.08 0.12 9.03 10.56 15.49 13.41 2.08 0.58 6.43

0.00 0.17 5.64 0.95 8.47 17.41 0.98 0.06 6.83 8.47 18.38 17.41 0.98 0.37 17.81

0.07 0.22 6.20 1.03 8.92 16.18 1.07 0.09 7.60 8.92 17.25 16.18 1.07 0.44 15.10

0.03 0.20 6.55 1.31 9.87 14.46 1.71 0.11 8.21 9.87 16.16 14.46 1.71 0.51 8.48

Note: 1 CON: control; MPO1: mixed plant oil 1; MPO2: mixed plant oil 2. 2 These values are analyzed. Saturated fatty acid (SFA) = C14: 0 + C16: 0 + C17: 0 + C18: 0 + C20: 0; Monounsaturated fatty acid (MUFA) = C16: 1n-7 + C18: 1n-9; Polyunsaturated fatty acid (PUFA) = C18: 2n-6 + C18: 3n-6 + C20: 4n-6 + C18: 3n-3 + C20: 5n-3 + C22: 6n-3; N-6 PUFA = C18: 2n-6 + C18: 3n-6 + C20: 4n-6; N-3 PUFA = C18: 3n-3 + C20: 5n-3 + C22: 6n-3. Table 4 Effects of mixed plant oils on performances of weanling piglets. Item

CON1

MPO11

MPO21

SEM

P-value

d d d d

8.77 12.74b 18.55b

8.80 13.43a 19.69a

8.79 13.21a 19.50a

0.01 0.14 0.25

0.89 < 0.01 0.02

284b 525 0.54b 2.83 3.54

331a 555 0.60a 2.80 1.01

315a 524 0.60a 2.76 1.01

9.45 16.41 0.04 0.05 1.05

0.02 0.36 < 0.01 0.62 0.26

414 762 0.55 2.77 1.80

448 782 0.58 2.73 1.00

450 772 0.58 2.71 1.03

14.65 26.43 0.05 0.04 0.85

0.22 0.86 0.42 0.61 0.40

349b 643 0.54b 2.80 2.67

389a 668 0.58a 2.77 1.00

383a 648 0.59a 2.74 1.02

8.93 17.11 0.04 0.04 0.95

0.02 0.56 0.05 0.53 0.35

0 body weight, kg 14 body weight, kg 28 body weight, kg 0 – 14 Average daily gain, g Average daily feed intake, g Feed efficiency Diarrhea score Diarrhea rate, % d 14 – 28 Average daily gain, g Average daily feed intake, g Feed efficiency Diarrhea score Diarrhea rate, % d 0 – 28 Average daily gain, g Average daily feed intake, g Feed efficiency Diarrhea score Diarrhea rate, %

Note: SEM means standard error of the mean. a-b Different superscripts within a row indicate a significant difference (P < 0.05). 1 CON: control; MPO1: mixed plant oil 1; MPO2: mixed plant oil 2.

3.3. The ATTD of nutrients Compared with CON, piglets fed diet supplemented with MPO1 and MPO2 had a higher (P < 0.05) ATTD of EE (Table 5), whereas these piglets did not show negative effects on ATTD of GE, CP and DM. 3.4. Serum biochemical indices Compared with CON, dietary supplementation with MPO1 showed increased (P < 0.05) concentration of TG on d 14 and an increased trend (P = 0.09) of concentration of TG on d 28 in piglets. Moreover, piglets fed MPO2 showed a higher (P < 0.05) concentration of GH on d 28 (Table 6). 5

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Table 5 Effects of mixed plant oils on nutrients digestibility of piglets on d 28 (%). Item

CON1

MPO11

MPO21

SEM

P-value

Gross energy Crude protein Dry matter Ether extract

85.40 80.90 84.76 69.12b

86.42 82.84 85.77 71.42a

85.61 81.39 85.31 71.14a

0.52 1.08 0.38 0.47

0.39 0.45 0.22 0.01

Note: SEM means standard error of the mean. a-b Different superscripts within a row indicate a significant difference (P < 0.05). 1 CON: control; MPO1: mixed plant oil 1; MPO2: mixed plant oil 2. Table 6 Effects of mixed plant oils on serum parameters of piglets. Item d 142 Urea, mmol/L TC, mmol/L TG, mmol/L TP, g/L GLU, mmol/L INS, μIU/mL COR, ng/mL GH, ng/mL d 282 Urea, mmol/L TC, mmol/L TG, mmol/L TP, g/L GLU, mmol/L INS, μIU/mL COR, ng/mL GH, ng/mL

CON1

MPO11

MPO21

SEM

P-value

5.05 3.19 0.48b 43.65 11.84 21.91 48.00 6.15

5.46 3.70 0.60a 47.05 11.35 25.63 53.01 6.35

4.77 3.22 0.47b 43.05 9.83 20.49 48.91 6.11

0.22 0.35 0.02 2.20 1.42 1.96 12.42 0.60

0.19 0.56 < 0.01 0.46 0.62 0.27 0.96 0.96

5.67 2.55 0.39 51.24 11.73 16.49 38.18 4.67b

4.82 2.96 0.55 49.59 9.71 17.92 47.34 3.77b

4.72 2.36 0.39 42.04 8.46 17.08 26.29 6.01a

0.91 0.30 0.05 3.94 1.87 1.04 6.65 0.26

0.74 0.42 0.09 0.32 0.52 0.65 0.20 < 0.01

Note: SEM means standard error of the mean. a-b Different superscripts within a row indicate a significant difference (P < 0.05). 1 CON: control; MPO1: mixed plant oil 1; MPO2: mixed plant oil 2. 2 TC: total cholesterol;TG: total glyceride; TP: total protein; GLU: glucose; INS: insulin; COR: cortisol; GH: growth hormone.

3.5. Serum immunity and antioxidant capacities Compared with CON, piglets fed MPO2 showed an increased concentration of IgM on d 14, and piglets fed MPO had increased (P < 0.05) concentration of IgG on d 28 (Table 6). There is an increased (P < 0.05) concentration of SOD and a decreased trend (P = 0.06) of MDA concentration in pigs fed MPO on d 14 in comparison with CON. Moreover, piglets fed MPO showed increased (P < 0.05) concentrations of SOD and GSH-Px, and piglets fed MPO2 showed higher (P < 0.05) concentration of CAT on d 28 in comparison with CON (Table 7). 3.6. Intestinal morphology Compared with CON, piglets fed MPO showed higher (P < 0.05) villus height in duodenum and jejunum. There are no negative effects of crypt depth and villus height / crypt depth in pigs fed MPO in comparison with CON (Table 8, Fig. 1). 4. Discussion After weaning, piglets need concentrated energy sources to meet and exceed the requirement of growth (Pettigrew et al., 1991). Therefore, better knowledge of the energy value of different energy feed combinations can help better understand the use of them in swine diets. In the present study, we utilized six different high quality plant oils to make novel mixed plant oils, we found the levels of SFA, MUFA, n-3 PUFA and n-3 / n-6 PUFA ratio were higher in MPO diet compared with SO diet, which is possibly due to that the linseed oil in MPO has high level of n-3 PUFA, whereas the palm oil and coconut oil contain large number of SFA. In current study, we also found that dietary supplementation with MPO showed improved ADG and FE in piglets compared with CON, which may be due to that a mixture of palm, soybean, and coconut oils could provide weanling piglets with easily digestible and absorbable essential fatty acids (Min et al., 2006). Another possible explanation may be that the MUFA (17.52% in MPO1 and 10.22% in MPO2) and PUFA (38.84% in MPO1 and 23.21% in MPO2) in diets provide piglets with more balanced fatty acid composition and energy to nitrogen 6

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Table 7 Effects of mixed plant oils on serum immunity and antioxidant capacity of piglets. Item d 142 IgA, g/L IgG, g/L IgM, g/L MDA, nmol/mL SOD, U/mL CAT, U/mL GSH-Px, U/mL d 282 IgA, g/L IgG, g/L IgM, g/L MDA, nmol/mL SOD, U/mL CAT, U/mL GSH-Px, U/mL

CON1

MPO11

MPO21

SEM

P-value

1.03 20.97 2.29b 3.70 75.35b 80.17 756.15

1.09 20.63 2.26b 3.88 86.08a 85.15 838.05

1.22 20.60 2.45a 4.30 83.31a 80.17 748.34

0.08 0.43 0.03 0.12 2.17 2.72 38.41

0.36 0.81 0.02 0.06 0.05 0.41 0.30

1.14 19.70b 2.62 5.36 82.61c 78.28b 665.21b

1.34 21.45a 2.48 4.15 105.54a 79.16ab 887.73a

1.24 21.62a 2.44 4.43 94.70b 83.61a 916.27a

0.07 0.42 0.10 0.38 2.46 1.14 12.43

0.28 0.05 0.46 0.17 < 0.01 0.05 < 0.01

Note: SEM means standard error of the mean. a-c Different superscripts within a row indicate a significant difference (P < 0.05). 1 CON: control; MPO1: mixed plant oil 1; MPO2: mixed plant oil 2. 2 CAT: catalase; GSH-Px: glutathione peroxidase; MDA: malondialdehyde; IgA: immunoglobulins A; IgG: immunoglobulins G; IgM: immunoglobulins M; SOD: superoxide dismutase. Table 8 Effects of mixed oils on intestinal morphology of piglets. Item Duodenum Villus height, μm Crypt depth, μm Villus height/Crypt depth Jejunum Villus height, μm Crypt depth, μm Villus height/Crypt depth Ileum Villus height, μm Crypt depth, μm Villus height/Crypt depth

CON1

MPO11

MPO21

SEM

P-value

373.39b 294.58 1.31

420.40a 275.19 1.61

445.92a 272.16 1.70

13.30 17.75 0.11

0.05 0.73 0.20

286.84b 184.86 1.68

307.79a 191.81 1.66

379.97a 215.89 1.84

13.26 23.87 0.20

0.05 0.73 0.87

261.82 148.81 1.81

241.50 162.46 1.56

231.95 142.69 1.72

26.44 12.40 0.11

0.88 0.63 0.52

Note: SEM means standard error of the mean. a-b Different superscripts within a row indicate a significant difference (P < 0.05). 1 CON: control; MPO1: mixed plant oil 1; MPO2: mixed plant oil 2.

ratio (Xu et al., 2016), which are demonstrated effective for the improvement of growth in piglets. Moreover, Dietary supplementation with functional fatty acids, such as ALA from linseed oil, can also improve nutrient digestibility and performance in piglets (Kim et al., 2007). Furthermore, the improved growth performance in piglets fed MPO2 may also be due to the positive effects of extruded corn on improving ADG and FE of early larvae after weaning (Lv et al., 2006). Besides, the addition of novel combination of six plant oils can supply a proper higher n-3 PUFA concentration and lower n-6 / n-3 PUFA ratios in diets for piglets compared with SO, which may also contribute the improvement of growth performance (ADG or FE) in animals (Long et al., 2018c; Li et al., 2015). Weanling piglets are easily to experience high diarrhea incidence, while in the current study the diarrhea rate in piglets fed SO or MPO was not very high, which may be due to the initial body weight of piglets is high and the energy supplementation meets or exceeds the requirement of piglets. The present study found the diarrhea rate of piglets fed MPO showed a reduction of 70%, 44% and 63% respectively in phase 1, 2 and overall compared with CON, which may be due to that the beneficial effect of n-3 PUFA in plant oils (such as flaxseed) on the improvement of gut microbiota structure and barrier function (Palla et al., 2015). However, the difference on diarrhea rate of piglets among all the treatments was not significant, while the study of Palla et al. (2015) showed significant reduction, these differences may be due to the variation of animals and composition of plant oils. The improvement of growth performance and reduction of diarrhea rate drive us suspect that the beneficial effects of dietary MPO supplementation may be largely related to the increase of nutrient digestibility and the improvement of antioxidant status, immune function as well as the intestinal development. To better understand the effects of MPO on the nutrient digestibility, we tested the ATTD of EE, GE, CP and DM. The present study showed the improved ATTD of EE in piglets fed MPO, which may be the reason that a proper lower dietary n-6 / n-3 PUFA ratio in piglets fed MPO can enhance the absorption and utilization of free amino acids and fatty acids (Li et al., 2015). Another explanation 7

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Fig. 1. Effects of mixed oils on intestinal morphology in duodenum, jejunum and ileum of piglets (T1: Villus height; T2: Crypt depth).

of the enhanced ATTD of EE might be that the medium chain fatty acid (MCFA) from the coconut and palm oils in MPO can supply piglets with instant energy and physiological benefits, such as expanding the spectrum of feed nutrients (Zentek et al., 2011). The MCFA can also be hydrolyzed by gastric and pancreatic lipases to provide rapid provision of energy for both enterocytes and intermediary hepatic metabolism in weanling piglets, which may strongly improve performance, immune function and intestinal health (Zentek et al., 2012). Besides, the MCFA (occur naturally as medium-chain triglycerides in coconut, soybean and palm oils) and PUFA (occur naturally as long-chain triglycerides in linseed oil) together may have some specific nutritional and metabolic effects, including rapid digestion, passive absorption and obligatory oxidation (Marten et al., 2006), thus enhancing the performance and ATTD of EE in weanling piglets. There are many biomarkers related to the growth performance, to better understand the reason for the improved performance, we estimated the GH, regular serum metabolite, and serum immune globulin and antioxidant status. In the present study, piglets fed diet supplemented with MPO2 showed a higher concentration of GH. Since GH is a peptide hormone that stimulates cell reproduction and regeneration, this result may highly related to the improved performance in piglets. The improved GH concentration may be due to that linseed oil in MPO can help prevent oxidative stress and enhance the expression of growth hormone related genes (Kutluyer et al., 2017). Moreover, dietary supplementation with MPO1 showed an increased concentration of TG in piglets, which is partly agreed with Min et al. (2006), who reported palm oil might increase the concentrations of cholesterol and triglyceride in weanling piglets. Another possible reason may be that MPO diets contain a high concentration of SFA, which might lead to a high concentration of plasma cholesterol (especially low density lipoprotein) (Gurr et al., 1989). The increased concentrations of immune globulins (IgM or IgG) in piglets fed diet supplemented with MPO reflect that MPO may improve the immune function and help piglets develop their own immune system, thus alleviating the weaning stress. The reason might be that n-3 PUFA in MPO diets could help modulate the weaning induced immune stress in piglets (Upadhaya et al., 2019). This 8

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result may also due to that reducing the dietary n-6: n-3 PUFA ratio can help improved plasma immunoglobulin and decreased the amount of cholesterol in serum, which may enhance the immune response in animals (Abdulla et al., 2019). Another possible reason for this results is mainly because of the MCFA and PUFA in MPO can enhance the lymphocyte proliferation in weanling piglets (Wang et al., 2006). Moreover, the MPO might also affect the local immune response in weanling by regulating the gut microbial ecosystem, which remained to be further investigated. Usually, the unsaturated fatty acids in some plant oils may make them prone to oxidation, which can damage some macromolecules, including DNA, proteins, and membrane lipids (Liang et al., 2015). However, the present study showed dietary MPO supplementation had improved levels of antioxidant enzymes, such as SOD, GSH-Px and CAT, indicating that MPO may play an important role in preventing endogenous lipids from peroxidation and oxidation, eliminating reactive free radicals (reactive oxygen species) and therefore alleviating oxidative stress response (Long et al., 2018b). Our results also showed that dietary MPO supplementation could also decrease the level of MDA, a major product of lipid peroxidation, indicating the antioxidant status of weanling piglets was improved. The reason for these positive effects on antioxidant capacities in weanling piglets may be due to the functional fatty acid, especially n-3 PUFA in linseed oil of MPO, can alleviate cell oxidation and improve intestinal health (Kim et al., 2007). Another reason of this result might be that MPO diets contain more concentrations of PUFA, which may impair oxidative status in piglets (Liu et al., 2014). The improved growth performance may also relate to the improvement of intestinal morphology. Current study showed the intestinal morphology, especially the villus height in duodenum and jejunum, was improved in piglets fed MPO, which probably reflects the improvement of nutrient digestion and absorption capacity of small intestine in piglets (Xiong et al., 2015). After weaning, epithelial structure and integrity of piglets are mainly affected by the sudden feed composition change and an increased rate of cell apoptosis (Van der Peet-Schwering et al., 2007). Therefore, weaning piglets may face severe reduction in villus height, which can decrease the ability to absorb nutrition since the intestinal villi are the site for nutrient absorption. The improved intestinal morphology may be the reason that MPO can decrease pathogenic bacteria in the gut of weanling piglets, which may help improve proliferation of epithelial cells to build villus (Mourao et al., 2005). Moreover, MPO may also improve intra-epithelial lymphocytes and local immune defense, and therefore improving the intestinal morphology in weanling piglets. Another explanation of our present results may be due to that MCFA in MPO can provide rapidly available energy for intestinal and extra-intestinal tissues in weanling piglets (Heo et al., 2002). Lee et al. (2007) also pointed out that MCFA could be utilized by enterocytes as energy feed and attenuate the negative effects of weaning on villus length and crypt depth in piglets. Additionally, previous studies suggest MCFA can also modulate the number of intra-epithelial lymphocytes, the lymphocyte proliferation rate and jejunal cytokine expression (Kuang et al., 2015), which may help improve the local immune system in weanling piglets. Besides, n-3 PUFA in MPO diets can help improve the functional properties of the gut morphology (Aziza et al., 2014), which may still remain to be further investigation. 5. Conclusion In conclusion, a mixed plant oils containing 10% coconut oil, 15% corn oil, 15% linseed oil, 15% peanut oil, 20% palm oil, and 25% soybean oil or these mixed plant oils combined with extruded corn can be used as better dietary energy feed than soybean oil in improving growth performance, serum immunity, antioxidant capacity, apparent total tract digestibility of ether extract and intestinal morphology in weanling piglets. Declaration of Competing Interest This work has no conflict of interest. Acknowledgements This research is supported by the National Natural Science Foundation of China (31772612) and China Agriculture Research System (CARS) 35. We thank Shandong Zhongda Agricultural Science and Technology Co., Ltd. for supplying the two novel mixed plant oils. References Association of Analytical Chemists, 2007. Official Methods of Analysis, 18th ed. AOAC Int., Gaithersburg, VA. Abdulla, N.R., Loh, T.C., Foo, H.L., Alshelmani, M.I., Akit, H., 2019. Influence of dietary ratios of n-6: n-3 fatty acid on gene expression, fatty acid profile in liver and breast muscle tissues, serum lipid profile, and immunoglobulin in broiler chickens. J. Appl. Poultry Res. 28, 454–469. Aziza, A.E., Awadin, W.F., Quezada, N., Cherian, G., 2014. Gastrointestinal morphology, fatty acid profile, and production performance of broiler chickens fed camelina meal or fish oil. Eur. J. Lipid Sci. Technol. 116, 1727–1733. Canakci, M., 2007. The potential of restaurant waste lipids as biodiesel feedstocks. Bioresour. Technol. 98, 183–190. Chen, X.L., Wang, N., Tian, M.L., Wang, L., Liu, T., Zhang, X.W., Shi, B.M., Shan, A.S., 2016. Dietary linseed oil in the maternal diet affects immunoglobulins, tissue fatty acid composition and expression of lipid metabolism-related genes in piglets. J. Anim. Physiol. Anim. Nutr. 101, 257–265. Ferrara, F., Tedin, L., Pieper, R., Meyer, W., Zentek, W., 2017. Influence of medium-chain fatty acids and short-chain organic acids on jejunal morphology and intraepithelial immune cells in weaned piglets. J. Anim. Physiol. Anim. Nutr. 101, 531–540. Gu, X.H., Li, D.F., 2003. Fat nutrition and metabolism in piglets: a review. Anim. Feed Sci. Technol. 109, 151–170. Gurr, M.I., Borlak, N., Ganatra, S., 1989. Dietary fat and plasma lipids. Nutr. Res. Rev. 2, 63–86. Heo, K.N., Lin, X., Han, I.K., Odle, J., 2002. Medium-chain fatty acids but not L-carnitine accelerate the kinetics of 14C triacylglycerol utilization by colostrumdeprived newborn pigs. J. Nutr. 132, 1989–1994.

9

Animal Feed Science and Technology xxx (xxxx) xxxx

S.F. Long, et al.

Kim, S.W., Mateo, R.D., Yin, Y.L., Wu, G.Y., 2007. Functional amino acids and fatty acids for enhancing production performance of sows and piglets. Asian-Australas. J. Anim. Sci. 20, 295–306. Kuang, Y., Wang, Y., Zhang, Y., Song, Y., Zhang, X., Lin, Y., Che, L., Xu, S., Wu, D., Xue, B., 2015. Effects of dietary combinations of organic acids and medium chain fatty acids as a replacement of zinc oxide on growth, digestibility and immunity of weaned pigs. Anim. Feed Sci. Technol. 208, 145–157. Kutluyer, F., Sirkecioğlu, A.N., Aksakal, E., Aksakal, F.İ., Tunç, A., Günaydin, E., 2017. Effect of dietary fish oil replacement with plants oils on growth performance and genes expression in juvenile rainbow trout (oncorhynchus mykiss). Annals. Anim. Sci. 17, 1135–1153. Lee, J., Mo, J.H., Katakura, K., Alkalay, I., Rucker, A.N., Liu, Y.T., Lee, H.K., Shen, C., Cojocatu, G., Shenouda, S., Kagnoff, M., Echmann, L., Ben-Neriah, Y., 2007. Maintenance of colonic homeostasis by distinctive apical TLR9 signaling in intestinal epithelial cells. Nat. Cell Biol. 8, 1327–1336. Lerma-Reyes, I., Mendoza-Martínez, G.D., Rojo-Rubio, R., Mejia, M., Garcia-Lopez, J.C., Lee-Rangel, H.A., 2018. Influence of supplemental canola or soybean oil on milk yield, fatty acid profile and postpartum weight changes in grazing dairy goats. Asian-Australas. J. Anim. Sci. 31, 225–229. Li, D.F., Thale, R.C., Nelssen, J.L., Harmon, D.L., Allee, G.L., Weeden, T.L., 1990. Effect of fat sources and combinations on starter pig performance, nutrient digestibility and intestinal morphology. J. Anim. Sci. 68, 3694–3704. Li, F., Duan, Y., Li, Y., Tang, Y., Geng, M., Oladele, O.A., Kim, S.W., Yin, Y., 2015. Effects of dietary n-6: n-3 PUFA ratio on fatty acid composition, free amino acid profile and gene expression of transporters in finishing pigs. Brit. J. Nutr. 113, 739–748. Liang, F., Jiang, S., Mo, Y., Zhou, G., Yang, L., 2015. Consumption of oxidized soybean oil increased intestinal oxidative stress and affected intestinal immune variables in yellow-feathered broilers. Asian-Australas. J. Anim. Sci. 28, 1194. Liu, P., Kerr, B.J., Weber, T.E., Chen, C., Johnston, L.J., Shurson, G.C., 2014. Influence of thermally oxidized vegetable oils and animal fats on intestinal barrier function and immune variables in young pigs. J. Anim. Sci. 92 (7), 2971–2979. Long, S.F., Xu, Y.T., Wang, C.L., Li, C.L., Liu, D.W., Piao, X.S., 2018a. Effects of dietary supplementation with a combination of plant oils on performance, meat quality and fatty acid deposition of broilers. Asian-Australas. J. Anim. Sci. 31, 1773–1780. Long, S.F., Xu, Y.T., Pan, L., Wang, Q.Q., Wang, C.L., Wu, J.Y., Wu, Y.Y., Han, Y.M., Yun, C.H., Piao, X.S., 2018b. Mixed organic acids as antibiotic substitutes improve performance, serum immunity, intestinal morphology and microbiota for weaned piglets. Anim. Feed Sci. Technol. 235, 23–32. Long, S.F., Kang, S., Wang, Q.Q., Xu, Y.T., Pan, L., Hu, J.X., Li, M., Piao, X.S., 2018c. Dietary supplementation with DHA-rich microalgae improves performance, serum composition, carcass trait, antioxidant status, and fatty acid profile of broilers. Poultry Sci. 97, 1881–1890. Lv, S., Li, D., Xing, J., Yongxi, M., Huang, D., Li, J., 2006. Effects of extrusion of corn on growth performance, nutrient digestibility and short-chain fatty acid profiles in the hindgut of weaned piglets. Arch. Anim. Nutr. 60, 170–179. Marten, B., Pfeuffer, M., Schrezenmeir, J., 2006. Medium-chain triglycerides. Int. Dairy J. 16, 1374–1382. Min, B.J., Kwon, O.S., Lee, W.B., Hong, J.W., Kim, I.H., 2006. The effect of soybean oil, dried palm oil powder and monoglyceride supplementation on growth performance and serum lipid changes in weaned pigs. J. Anim. Sci. Technol. 48, 203–210. Mourao, J.L., Pinheiro, V., Alves, A., Guedes, C.M., Pinto, L., Saavedra, M.J., Spring, P., Kocher, A., 2005. Effect of mannan oligosaccharides on the performance, intestinal morphology and cecal fermentation of fattening rabbits. Anim. Feed Sci. Technol. 126, 107–120. NRC, 2012. Nutrient Requirements of Swine, 11th rev. ed. Natl. Acad. Press, Washington, DC, USA. Pettigrew, J.E., Moser, R.L., Miller, E.R., Ullrey, D.E., Lewis, A.J., 1991. Fat in swine nutrition. Swine nutrition 133–145. Rossi, R., Pastorelli, G., Cannata, S., Corino, C., 2010. Recent advances in the use of fatty acids as supplements in pig diets: a review. Anim. Feed Sci. Technol. 162, 1–11. Teoh, C.Y., Ng, W.K., 2016. The implications of substituting dietary fish oil with vegetable oils on the growth performance, fillet fatty acid profile and modulation of the fatty acid elongase, desaturase and oxidation activities of red hybrid tilapia, Oreochromis sp. Aquaculture 465, 311–322. Upadhaya, S.D., Yun, H.M., Lee, K.Y., Kim, I.H., 2019. Do varied dietary omega-6 to omega-3 ratios affect the performance, nutrient digestibility, immune status and faecal microbiota of weaner pigs? Anim. Prod. Sci. 59, 236–242. Van der Peet-Schwering, C.M.C., Jansman, A.J.M., Smidt, H., Yoon, I., 2007. Effects of yeast culture on performance, gut integrity, and blood cell composition of weaning pigs. J. Anim. Sci. 85, 3099–3109. Vilarrasa, E., Barroeta, A.C., Tres, A., Estevegarcia, E., 2015. Use of re-esterified palm oils, differing in their acylglycerol structure, in weaning-piglet diets. Animal. 9, 1–8. Wang, J., Wu, X., Simonavicius, N., Tian, H., Ling, L., 2006. Medium-chain fatty acids as ligands for orphan G proteincoupled receptor GPR84. J. Bio. Chem. 28, 34457–34464. Wiseman, J., Cole, D.J.A., Hardy, B., 1990. The dietary energy values of soya-bean oil, tallow and their blends for growing/finishing pigs. Anim. Prod. 50, 513–518. Xiong, X., Yang, H.S., Wang, X.C., Hu, Q., Liu, C.X., Wu, X., Deng, D., Hou, Y.Q., Nyachoti, C.M., Xiao, D.F., 2015. Eff ;ect of low dosage of chito-oligosaccharide supplementation on intestinal morphology, immune response, antioxidant capacity, and barrier function in weaned piglets. J. Anim. Sci. 93, 1089–1097. Xu, X., Wang, H.L., Piao, X.S., 2016. Validation of metabolisable energy prediction equation for de-oiled corn distillers dried grains with solubles fed to finishing pigs. Italian J. Anim. Sci. 15, 55–61. Xu, Y.T., Ma, X.K., Wang, C.L., Yuan, M.F., Piao, X.S., 2018. Effects of dietary valine: lysine ratio on the performance, amino acid composition of tissues and mRNA expression of genes involved in branched-chain amino acid metabolism of weaned piglets. Asian-Australas. J. Anim. Sci. 31, 106–115. Zentek, J., Buchheitrenko, S., Ferrara, F., Vahjen, W., van Kessel, A.G., Pieper, R., 2011. Nutritional and physiological role of medium-chain triglycerides and mediumchain fatty acids in piglets. Anim. Health Res. Rev. 12, 83–93. Zentek, J., Buchheit-Renko, S., Männer, K., Pieper, R., Vahjen, W., 2012. Intestinal concentrations of free and encapsulated dietary medium-chain fatty acids and effects on gastric microbial ecology and bacterial metabolic products in the digestive tract of piglets. Arch. Anim. Nutr. 66, 14–26.

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