Effect of different ratios of phytogenic feed additives on growth performance, nutrient digestibility, intestinal barrier integrity, and immune response in weaned pigs challenged with a pathogenic Escherichia coli

Abstract

This study was conducted to investigate the effects of supplementing different ratios of phytogenic feed additives (PFA) to weaned pigs challenged with pathogenic Escherichia coli on growth performance, nutrient digestibility, intestinal barrier integrity, and immune response, and to determine the optimal mixing ratio for post-weaning diarrhea (PWD) prevention. A total of 48 4-wk-old weaned pigs with initial body weight of 8.01 ± 0.39 kg were placed in individual metabolic cages, and then randomly assigned to eight treatment groups. The eight treatments were as follows: a basal diet without E. coli challenge (negative control, NC), a basal diet with E. coli challenge (positive control, PC), PC with supplementing 0.1% mixture of 20% bitter citrus extract (BCE), 10% microencapsulated blend of thymol and carvacrol (MEO), and 70% excipient (T1), PC with supplementing 0.1% mixture of 10% MEO, 20% premixture of grape seed and grape marc extract, green tea, and hops (PGE), and 60% excipient (T2), PC with supplementing 0.1% mixture of 10% BCE, 10% MEO, 10% PGE, and 70% excipient (T3), PC with supplementing 0.1% mixture of 20% BCE, 20% MEO, and 60% excipient (T4), PC with supplementing 0.1% mixture of 20% MEO, 20% PGE, and 60% excipient (T5), and PC with supplementing 0.1% mixture of 10% BCE, 20% MEO, 10% PGE, and 60% excipient (T6). The experiments progressed in 16 days, including 5 days before and 11 days after the first E. coli challenge (day 0). In the E. coli challenge treatments, all pigs were orally inoculated by dividing a total of 10 mL of E. coli F 18 for three consecutive days from day 0 postinoculation (PI). Compared with the PC group, the PFA2 and PFA6 groups significantly increased (P < 0.05) feed efficiency and decreased (P < 0.05) diarrhea during the entire period. At day 11 PI, the PFA6 group significantly improved (P < 0.05) gross energy digestibility compared to the PFA1 group. The PFA6 group significantly decreased (P < 0.05) tumor necrosis factor α (TNF-α) and interleukin-6 in serum and increased (P < 0.05) the villus height to crypt depth ratio (VH:CD). The PFA2 significantly decreased (P < 0.05) the relative protein expression of calprotectin in the ileum. In conclusion, improvements in growth performance, diarrhea reduction, and immunity enhancement are demonstrated when 10% BCE, 20% MEO, 10% PGE, and 60% excipient are mixed.

Phytogenic feed additives (PFA) are considered an alternative to antibiotics in the swine industry through their antibacterial and antioxidant action, and studies are being actively conducted to prevent the decrease in growth performance and immune response due to post-weaning diarrhea caused by pathogenic Escherichia coli using PFA. Among the six PFA mixing ratios used in this study, supplementing a mixture of 10% bitter citrus extract, 20% microencapsulated blend of thymol and carvacrol, 10% premixture of grape seed and grape marc extract, green tea, and hops, and 60% excipient improved growth performance, the immune response, and the intestinal morphology of weaned pigs.

Introduction

Phytogenic feed additives (PFA) include various herbs and spices, such as essential oils and polyphenols (Ahmed et al., 2013). The PFA contain a wide range of bioactive compounds with beneficial health effects (Upadhaya and Kim, 2017). The beneficial health effects of PFA in animals are related to alterations in intestinal microbiota, the increased absorbance of nutrients, and anti-oxidative and immunomodulatory activities (Jiang et al., 2014; Caprarulo et al., 2022). Flavonoids and polyphenols contained in PFA are generally known to have antioxidant and antibacterial actions and based on this, the PFA is considered an alternative to antibiotics in the swine industry (Montoya et al., 2021).

Pathogenic Escherichia coli (E. coli) infection is one of the most important causes of post-weaning diarrhea (PWD) in pigs (Oh et al., 2021). The PWD caused by E. coli infection decreases intestinal villus height (VH) and increases intestinal cellular permeability and pro-inflammatory cytokines (He et al., 2021; Song et al., 2022). Intestinal damage causes severe diarrhea and leads to poor growth performance and the mortality of weaned pigs, resulting in significant financial loss to the swine industry (Bonetti et al., 2021; Yi et al., 2021).

In a previous study, we showed that diarrhea was reduced when thymol and carvacrol were added to weaned pigs challenged with E. coli (Chang et al., 2022). Feeding a premixture of grape seed and grape marc extract, green tea, and hops (PGE), containing more than 10% of flavonoids, the level of tumor necrosis factor α (TNF-α), a pro-inflammatory cytokine in the blood, decreased and showed an anti-inflammatory effect. Also, improvements in immune response and intestinal morphology were observed when feeding a mixture of bitter citrus extract (BCE), thymol and carvacrol at a mixing ratio of 4:5. Accordingly, we hypothesized that the effect of each PFA could be maximized through a synergistic effect when the PFA was fed in combination rather than as a single agent. Also, it is expected to reduce the economic burden of farms by reducing the cost of relatively high unit prices of additives. However, studies on mixed PFA in different ratios are not sufficient, and additional studies are needed to determine the optimal ratio of mixed PFA effective for PWD. To determine the optimal ratio of mixed PFA, three PFA that showed effects on PWD alleviation in our previous studies were selected. Therefore, this study investigated the effects of supplementation with different ratios of PFA to weaned pigs challenged with pathogenic E. coli on growth performance, nutrient digestibility, intestinal barrier integrity, and the immune response, and determined the optimal mixing ratio for PWD prevention.

Materials and Methods

The protocol for this study was reviewed and approved by the Institutional Animal Care and Use Committee of Chungbuk National University, Cheongju, Korea (approval no. CBNUA-1698-22-02).

Preparation of phytogenic feed additives

In this study, six types of PFA were fed (Table 1). The PFA1 is a mixture of 20% BCE, 10% microencapsulated blend of thymol and carvacrol (MEO), and 70% excipient. The PFA2 is a mixture of 10% MEO, 20% PGE, and 60% excipient. The PFA3 is a mixture of 10% BCE, 10% MEO, 10% PGE, and 70% excipient. The PFA4 is a mixture of 20% BCE, 20% MEO, and 60% excipient. The PFA5 is a mixture of 20% MEO, 20% PGE, and 60% excipient. The PFA6 is a mixture of 10% BCE, 20% MEO, 10% PGE, and 60% excipient. The BCE (BioFlavex GC, HTBA, Beniel, Spain) contains 25% to 27% naringin and 11% to 15% neohesperidin. The MEO (Avipower 2, VetAgro SpA, Reggio, Emmilia, Italy) contains 7% of thymol and 7% of carvacrol. The PGE (AntaOx Flavosyn, DR. Eckel GmbH, Niederzissen, Germany) contains more than 10% of flavonoids. All PFA using this study were obtained by a commercial company (Eugene-Bio, Suwon, South Korea).

Table 1.

Specification of phytogenic feed additives (PFA) used in this study

Items, %	PFA1	PFA2	PFA3	PFA4	PFA5	PFA6
Bitter citrus extract1	20	—	10	20	—	10
Microencapsulated blend of thymol and carvacrol2	10	10	10	20	20	20
Premixture of grape seed and grape marc extract, green tea, and hops3	—	20	10	—	20	10
Excipient	70	70	70	60	60	60
Total	100	100	100	100	100	100

¹BioFlavex GC, HTBA, Beniel, Spain.

²Avipower 2, VetAgro SpA Reggio, Emmilia, Italy.

³AntaOx Flavosyn, DR. Eckel GmbH, Niederzissen, Germany.

Animals, treatments and experimental design

A total of 48 4-wk-old crossbred weanling pigs ([Landrace × Yorkshire] × Duroc) with an initial body weight (BW) of 8.01 ± 0.39 kg were used in this study. All pigs were assigned to a completely randomized into eight treatment groups based on the initial BW. There was one pig treatment in a cage and six replicate cages per treatment. Pigs were individually placed in 45 × 55 × 45 stainless steel metabolism cages in an environmentally controlled room. Pigs were housed in individual pens for 16 days, including 5 days before and 11 days after the first E. coli challenge (day 0). The dietary treatments were as follows: a basal diet without E. coli challenge (negative control, NC), a basal diet with E. coli challenge (positive control, PC), PC with supplementing 0.1% PFA1 (T1), PC with supplementing 0.1% PFA2 (T2), PC with supplementing 0.1% PFA3 (T3), PC with supplementing 0.1% PFA4 (T4), PC with supplementing 0.1% PFA5 (T5), and PC with supplementing 0.1% PFA6 (T6). All diets were formulated to meet or exceed the NRC (2012) requirement (Table 2). All treatment groups were fed the experimental diet during the entire experimental period including the adaptation period. The diets were mixed with water in a 1:1 ratio before feeding and were fed at 08:30 a.m. and 5:30 p.m. each day. The pigs had ad libitum access to water.

Table 2.

Compositions of basal diets (as-fed-basis)¹

Items	Content
Ingredients, %
Corn	34.43
Extruded corn	15.00
Lactose	10.00
Dehulled soybean meal, 51% CP1	13.50
Soy protein concentrate, 65% CP1	10.00
Plasma powder	6.00
Whey	5.00
Soy oil	2.20
Monocalcium phosphate	1.26
Limestone	1.40
l-Lysine-HCl, 78%	0.06
dl-Methionine, 50%	0.15
Choline chloride, 25%	0.10
Vitamin premix2	0.25
Trace mineral premix3	0.25
NaCl	0.40
Total	100.00
Calculated value
ME, kcal/kg	3433
CP, %	20.76
Lysine, %	1.35
Methionine, %	0.39
Ca	0.82
P	0.65
Analyzed value
ME, kcal/kg	3512
CP, %	20.92

¹Abbreviation: CP, crude protein; ME, metabolize energy.

²Provided per kilogram of complete diet: vitamin A, 11,025 IU; vitamin D₃, 1,103 IU; vitamin E, 44 IU; vitamin K₃, 4.4 mg; riboflavin, 8.3 mg; niacin, 50 mg; thiamine, 4 mg; d-pantothenic, 29 mg; and vitamin B₁₂, 33 mg.

³Provided per kilogram of complete diet without Zinc: Cu (as CuSO₄•5H₂O), 12 mg; Mn (as MnO₂), 8 mg; I (as KI), 0.28 mg; and Se (as Na₂SeO₃•5H₂O), 0.15 mg.

Bacterial strains and culture

Shiga toxin-producing E. coli F18 was provided in stock form. The F18 E. coli expressed heat labile toxin (LT) and shiga toxin type 2e. Ten microliters of thawed E. coli stock were inoculated into 10 mL of nutrient broth and cultured at 37 °C for 18 to 24 h, and then subcultured. Thereafter, the subcultured E. coli was smeared on MacConkey agar to confirm the bacterial enumeration. A final concentration of 1.0 × 10¹⁰ CFU/mL was used in this study. In 5 d after adaptation, all pigs except NC group pigs were orally challenged by a total of 10 mL of E. coli F18 by dividing for three consecutive days from day 0 post-inoculation (PI).

Growth performance and diarrhea scores

All pigs were individually weighed at the initial (day −5), days 0, 7, and 11 PI. Feed intake was documented to calculate the average daily feed intake (ADFI) and feed efficiency (G:F). The average daily gain (ADG), ADFI, and G:F were calculated for each period (days −5 to 0, days 0 to 7 PI, days 7 to 11 PI, and days 0 to 11 PI). The diarrhea scores were individually recorded at 08:00 a.m. and 5:00 p.m. by the same person during the entire experimental period. The diarrhea scores were as follows: 0 (normal feces), 1 (soft feces), 2 (mild diarrhea), and 3 (severe diarrhea). Scores were calculated as the average diarrhea score for each period per treatment group by summing the average daily diarrhea scores of each pig. The frequency of diarrhea was calculated by counting pen days in which the average diarrhea score of individual pigs in each pen was ≥ 2.

Nutrient digestibility

Apparent total tract digestibility (ATTD) of dry matter (DM), crude protein (CP), and gross energy (GE) were determined using chromic oxide (0.2%) as an inert indicator by Fenton and Fenton (1979) method. Pigs were fed diets mixed with chromic oxide from days 3 to 4, and 7 to 8. Fresh excreta samples were collected from six pigs per treatment (days 5 and 9). At the same time, those diet samples were also collected. Fresh fecal and diet samples were stored in a freezer at −20 °C immediately after collection. At the end of the experiment, fecal samples were dried at 70 °C for 72 h and then crushed on a 1 mm screen. All diet and fecal samples were then analyzed for DM, CP, and GE following the procedures by the AOAC (2007). Chromium levels were determined via UV absorption spectrophotometry (UV-1201, Shimadzu, Kyoto, Japan) using Williams et al. (1962) method. The GE of diets and feces were analyzed using an adiabatic oxygen bomb calorimeter (6400 Automatic Isoperibol calorimeter, Parr, USA). For calculating the ATTD of the nutrients, we used the following equation: Digestibility = 1 − [(Nf × Cd)/(Nd × Cf)] × 100, where Nf = concentration of nutrient in fecal, Nd = concentration of nutrient in the diet, Cd = concentration of chromium in the diet, and Cf = concentration of chromium in the fecal.

Blood profiles

Blood samples were collected from the jugular vein of all pigs before E. coli challenge (day 0) and on days 2, 4, 7, and 11 PI. At the time of collection, blood samples were collected into vacuum tubes containing K₃EDTA for complete blood count analysis, and nonheparinized tubes for serum analysis, respectively. After collection, serum samples were centrifuged at 3,000 × g for 20 min at 4 °C. The white blood cell (WBC), basophil, neutrophil, and lymphocyte levels in the whole blood were measured using an automatic blood analyzer (ADVIA 120, Bayer, NY, USA). Immunoglobulin G (IgG) and immunoglobulin A (IgA) levels were gauged using an automatic biochemistry blood analyzer (Hitachi 747; Hitachi, Tokyo, Japan). The pro-inflammatory cytokine such as interleukin-6 (IL-6) and TNF-α was measured using commercially available ELISA kits (Quantikine, R&D systems, Minneapolis, MN, USA) and the absorbance was measured at 450 nm.

Intestinal morphology and histology analysis

At the end of the experiment (day 11 PI), all pigs (1 treatment/6 pigs) were anesthetized with carbon dioxide gas after blood sampling and euthanized by exsanguination. After euthanization, intestinal tissues of about 10 cm from the ileum (close to the ileocecal junction) were collected. Among the collected ­intestine samples, samples for intestinal morphology and goblet cell analysis were washed with 10% neutral buffered formalin (Sigma-Aldrich, St. Louis, MO, USA), fixed, and stored refrigerated until staining for ­analysis. ­Samples for analysis of tight junction protein were stored at −80 °C from immediately after collection until analysis. The fixed intestinal segments were dehydrated and embedded in paraffin. Cross sections of a thickness of 5 µm were sectioned and stained with hematoxylin and eosin (H&E) for measuring intestinal morphology. For the counting of goblet cell numbers, sections were stained with a combination of alcian blue and periodic acid-Schiff. The slides were examined using an Olympus IX51 inverted phase-contrast microscope. Intestinal morphological measurements of VH, crypt depth (CD), and villus height to crypt depth ratio (VH:CD) were calculated for the mean value of six well-orientated villus and crypts, respectively. Goblet cells were counted in the selected six villus and crypts.

Expression of tight junction proteins

The intestinal sample stored at −80 °C after sampling was homogenized and used for calprotectin and claudin 1 (CLDN1) concentration analysis. The concentration of total protein was quantified using a Pierce BCA protein assay kit (#23225, Thermo Fisher Scientific, Waltham, MA, USA). After the homogenized intestinal sample was diluted to reach a working range of 20 to 2,000 μg/mL, the absorbance was measured at 562 nm. The total protein concentration was calculated as a standard curve and used to normalize the concentrations of calprotectin and CLDN1. The relative protein expression of calprotectin and CLDN1 was ­determined by using commercially available ELISA kits (Cat no. MBS707210, MBS025129; Mybiosource, San Diego, CA, USA). Homogenized intestinal samples were diluted to reach a working range of 0.312 to 20 ng/mL for calprotectin and 0.5 to 16 ng/mL for CLDN1. Both absorbances were measured at 450 nm. The concentrations of calprotectin and CLDN1 were calculated by the standard curve and described as ng/mg of protein.

Statistical analysis

All data except for the frequency of diarrhea were analyzed via the general linear model procedures of SAS (SAS Institute, Cary, NC, USA), using each pen as the experimental unit. The frequency of diarrhea was compared with a chi-square test, using the FREQ procedure of SAS. The frequency of diarrhea was visualized using GraphPad Prism 8 software (GraphPad Software, San Diego, CA, USA). Differences between treatment means were determined using Tukey’s multiple range test. Variability in the data was expressed as the pooled standard error. A probability level of P < 0.05 was indicated to be statistically significant, and a level of 0.05 ≤ P < 0.10 was considered to have such a tendency.

Results

Growth performance

There was no difference in BW among treatment groups before the E. coli challenge (Table 3). On both days 7 and 11 PI, the PC group had significantly lower (P < 0.05) BW than the NC group. The T4 and T6 groups had significantly higher (P < 0.05) BW than the PC group at the same period. At days 0 to 7 PI, the T4 and T6 groups had significantly higher (P < 0.05) ADG and G:F than the PC group. At days 7 to 11 PI, the T2 group had significantly higher (P < 0.05) ADG and G:F than the PC group. During the entire period, the T6 group significantly improved (P < 0.05) in all of the ADG, ADFI, and G:F compared to the PC group.

Table 3.

Effect of different ratios of phytogenic feed additives on growth performance of weaned pigs challenged with Escherichia coli¹

Items	NC	PC	T1	T2	T3	T4	T5	T6	SE2	P-value
BW, kg
Day −5	8.01	8.00	8.03	8.01	8.00	8.02	7.99	7.98	0.176	1.000
Day 0	8.36	8.38	8.45	8.23	8.29	8.36	8.24	8.28	0.167	0.984
Day 7 PI	9.97ab	9.28c	9.38bc	9.77abc	9.46bc	10.15a	9.39bc	10.01ab	0.145	<0.001
Day 11 PI	11.02ab	10.21c	10.58abc	11.06ab	10.49bc	11.09ab	10.63abc	11.24a	0.151	<0.001
Days −5 to 0
ADG, g	69.33	74.67	83.33	42.67	59.33	67.33	51.33	60.00	12.911	0.439
ADFI, g	126.67bc	135.33bc	193.33a	112.00c	124.67bc	156.00abc	164.00ab	144.67abc	11.424	<0.001
G:F, g/g	0.55	0.52	0.41	0.40	0.47	0.43	0.31	0.38	0.077	0.451
Days 0 to 7 PI
ADG, g	229.52a	128.57b	133.81b	220.95ab	166.67ab	255.71a	163.33ab	246.67a	20.884	<0.001
ADFI, g	344.76a	325.95abc	318.57c	333.81abc	323.10bc	340.00ab	343.33ab	343.33ab	4.635	<0.001
G:F, g/g	0.67abc	0.40c	0.41c	0.66abc	0.51abc	0.75a	0.48bc	0.72ab	0.060	<0.001
Days 7 to 11 PI
ADG, g	262.50abc	233.33c	299.17abc	320.83a	256.67abc	236.67bc	311.67ab	308.33abc	16.762	0.001
ADFI, g	397.50a	380.83b	397.50a	392.08ab	398.33a	400.00a	395.00ab	400.00a	3.662	0.012
G:F, g/g	0.66abc	0.61bc	0.75abc	0.82a	0.64abc	0.59c	0.79ab	0.77abc	0.042	0.001
Days 0 to 11 PI
ADG, g	241.51ab	166.67c	193.94bc	257.27ab	199.39abc	248.79ab	217.27abc	269.09a	15.811	<0.001
ADFI, g	363.94a	345.91b	347.28b	355.00ab	350.46ab	361.82ab	362.12ab	363.94a	3.683	0.001
G:F, g/g	0.66ab	0.48b	0.55ab	0.72a	0.57ab	0.69a	0.60ab	0.74a	0.041	0.001

¹Abbreviation: NC, basal diet without E. coli challenge (negative control); PC, basal diet with E. coli challenge (positive control); T1, PC + PFA1 0.1%; T2, PC + PFA2 0.1%; T3, PC + PFA3 0.1%; T4, PC + PFA4 0.1%; T5, PC + PFA5 0.1%; T6, PC + PFA6 0.1%; BW, body weight; PI, post-inoculation; ADG, average daily gain; ADFI, average daily feed intake; G:F, feed efficiency; SE, standard error.

² n = 6 per treatment.

^a-cMeans with different letters are significantly differ (P < 0.05).

Diarrhea score

Before E. coli challenge, there was no difference in the diarrhea score among treatment groups (Table 4). After E. coli challenge (days 0 to 7 PI and days 0 to 11 PI), the PC group significantly increased (P < 0.05) diarrhea score and frequency of diarrhea compared to the NC group (Fig. 1). At days 7 to 11 PI, the T5 and T6 groups had significantly decreased (P < 0.05) the diarrhea score compared to the PC group. The T2 and T6 groups had significantly alleviated (P < 0.05) the diarrhea score compared to the PC group during the entire period.

Table 4.

Effect of different ratios of phytogenic feed additives on diarrhea score of weaned pigs challenged with Escherichia coli¹

Items	NC	PC	T1	T2	T3	T4	T5	T6	SE2	P-value
Diarrhea score3
Days −5 to 0	1.42	1.50	1.17	1.25	1.38	1.71	1.67	1.17	0.203	0.399
Days 0 to 7 PI	1.03b	2.05a	1.76a	1.43ab	1.55ab	1.62ab	1.57ab	1.38ab	0.157	0.005
Days 7 to 11 PI	0.57ab	1.03a	0.73ab	0.60ab	0.67ab	0.63ab	0.50b	0.47b	0.108	0.022
Days 0 to 11 PI	0.83b	1.63a	1.33ab	1.08b	1.18ab	1.21ab	1.13ab	1.00b	0.117	0.002

¹Abbreviation: NC, basal diet without E. coli challenge (negative control); PC, basal diet with E. coli challenge (positive control); T1, PC + PFA1 0.1%; T2, PC + PFA2 0.1%; T3, PC + PFA3 0.1%; T4, PC + PFA4 0.1%; T5, PC + PFA5 0.1%; T6, PC + PFA6 0.1%; PI, post-inoculation; SE, standard error.

² n = 6 per treatment.

³Diarrhea score was determined as follow: 0, normal feces; 1, soft feces; 2, mild diarrhea; 3, severe diarrhea.

^a,bMeans with different letters are significantly differ (P < 0.05).

Figure 1.

Effect of different ratios of phytogenic feed additives on frequency of diarrhea of weaned pigs challenged with Escherichia coli. NC, basal diet without E. coli challenge (negative control); PC, basal diet with E. coli challenge (positive control); T1, PC + PFA1 0.1%; T2, PC + PFA2 0.1%; T3, PC + PFA3 0.1%; T4, PC + PFA4 0.1%; T5, PC + PFA5 0.1%; T6, PC + PFA6 0.1%; Each value is the mean value of 6 replicates. Frequency of diarrhea = (number of pigs with diarrhea/number of pen days) × 100.

Nutrient digestibility

At day 7 PI, the PC and T1 groups had significantly lower (P < 0.05) DM digestibility than the NC group (Table 5). At day 11 PI, the T6 group significantly improved (P < 0.05) GE digestibility compared to the T1 group. There were no differences in DM and CP digestibility among treatment groups at day 11 PI.

Table 5.

Effect of different ratios of phytogenic feed additives on nutrient digestibility of weaned pigs challenged with Escherichia coli¹

Items	NC	PC	T1	T2	T3	T4	T5	T6	SE2	P-value
Day 7 PI
DM, %	80.12a	76.89b	77.10b	78.01ab	77.43ab	78.37ab	77.83ab	77.95ab	0.656	0.043
CP, %	72.22	69.61	69.81	71.64	71.22	70.44	71.18	71.46	1.292	0.829
GE, %	73.06	71.36	71.13	72.21	73.23	72.11	74.55	73.84	1.158	0.406
Day 11 PI
DM, %	81.16	77.65	78.09	79.63	78.22	78.83	78.38	80.35	0.919	0.119
CP, %	75.17	73.86	74.25	74.83	74.95	74.17	74.73	75.02	0.755	0.905
GE, %	74.21ab	71.91ab	70.22b	72.61ab	73.29ab	70.79ab	71.76ab	76.01a	1.195	0.033

¹Abbreviation: NC, basal diet without E. coli challenge (negative control); PC, basal diet with E. coli challenge (positive control); T1, PC + PFA1 0.1%; T2, PC + PFA2 0.1%; T3, PC + PFA3 0.1%; T4, PC + PFA4 0.1%; T5, PC + PFA5 0.1%; T6, PC + PFA6 0.1%; DM, dry matter; CP, crude protein; GE, gross energy; PI, post-inoculation; SE, standard error.

² n = 6 per treatment.

^a,bMeans with different letters are significantly differ (P < 0.05).

Blood profile

Before E. coli challenge, there were no differences in WBC, basophil, neutrophil, and lymphocyte levels among treatment groups (Table 6). At day 2 PI, the WBC levels had significantly decreased (P < 0.05) in the T2 and T3 groups compared to the T1 group. The T2 group had significantly lower (P < 0.05) WBC levels than the PC group at day 4 PI. In the same period, compared to the PC and the PFA-added treatment groups, the T2 group showed a low tendency (P = 0.064) of neutrophil levels and a high tendency (P = 0.076) of lymphocyte levels. At day 7 PI, the T2 group had significantly decreased (P < 0.05) basophil levels compared to the PC group. In the same period, the PC group had significantly lower (P < 0.05) lymphocyte levels than the NC group. Also, the PFA-added treatment groups except for the T2 and T5 groups had significantly increased (P < 0.05) lymphocyte levels compared to the PC group. At day 11 PI, the T4 group had significantly higher (P < 0.05) lymphocyte levels compared to the PC group.

Table 6.

Effect of different ratios of phytogenic feed additives on blood profile of weaned pigs challenged with Escherichia coli¹

Items	NC	PC	T1	T2	T3	T4	T5	T6	SE2	P-value
Day 0
WBC, 103/µL	18.25	19.54	18.46	13.78	14.97	15.61	15.25	14.18	1.765	0.190
Basophil, %	0.13	0.13	0.10	0.17	0.13	0.13	0.10	0.17	0.032	0.724
Neutrophil, %	40.20	49.67	46.63	41.57	47.73	40.87	45.00	43.10	3.433	0.434
Lymphocyte, %	54.53	43.93	43.40	53.17	47.60	53.70	44.50	46.20	3.720	0.166
Day 2 PI
WBC, 103/µL	23.44ab	25.58ab	27.80a	16.12b	16.38b	18.85ab	19.84ab	18.83ab	2.292	0.005
Basophil, %	0.03ab	0.10a	0.03ab	0.07ab	0.10a	0.03ab	0.10a	0.00b	0.020	0.003
Neutrophil, %	43.87b	55.00ab	50.60ab	57.70a	49.23ab	49.53ab	58.17a	52.23ab	2.899	0.020
Lymphocyte, %	47.93a	35.23b	42.57ab	36.67b	42.30ab	44.27ab	36.27b	40.63ab	2.839	0.036
Day 4 PI
WBC, 103/µL	21.22abc	29.25a	28.15ab	18.09c	21.19abc	21.39abc	19.04bc	22.03abc	2.196	0.006
Basophil, %	0.03	0.07	0.03	0.07	0.07	0.07	0.10	0.07	0.027	0.727
Neutrophil, %	47.67	62.53	47.40	45.90	50.30	54.23	54.30	52.83	3.688	0.064
Lymphocyte, %	45.37	30.47	47.33	49.03	38.43	40.20	41.60	42.40	4.102	0.076
Day 7 PI
WBC, 103/µL	20.41	26.13	23.95	18.33	21.25	18.41	20.60	23.04	2.193	0.186
Basophil, %	0.13ab	0.20a	0.10ab	0.03b	0.13ab	0.13ab	0.03b	0.13ab	0.034	0.022
Neutrophil, %	38.27abc	48.17a	21.00d	39.50ab	23.87cd	31.60bcd	34.83abcd	41.37ab	3.447	<0.001
Lymphocyte, %	52.37a	30.67b	59.77a	46.73ab	55.77a	58.37a	45.27ab	53.50a	4.265	0.001
Day 11 PI
WBC, 103/µL	21.38	21.77	23.03	24.11	19.64	19.79	20.37	22.58	1.406	0.274
Basophil, %	0.03	0.03	0.03	0.07	0.07	0.03	0.13	0.03	0.025	0.073
Neutrophil, %	34.20b	49.23a	43.27ab	44.37ab	43.30ab	36.90ab	45.33ab	41.40ab	2.755	0.013
Lymphocyte, %	48.13ab	37.10b	52.53ab	51.03ab	39.77b	59.07a	46.83ab	53.93ab	4.093	0.009

¹Abbreviation: NC, basal diet without E. coli challenge (negative control); PC, basal diet with E. coli challenge (positive control); T1, PC + PFA1 0.1%; T2, PC + PFA2 0.1%; T3, PC + PFA3 0.1%; T4, PC + PFA4 0.1%; T5, PC + PFA5 0.1%; T6, PC + PFA6 0.1%; WBC, white blood cell; PI, post-inoculation; SE, standard error.

² n = 6 per treatment.

^a-^dMeans with different letters are significantly differ (P < 0.05).

The levels of IgG, IgA, TNF-α, and IL-6 in serum before E. coli challenge were no differences among treatment groups (Table 7). At day 2 and 4 PI, the PC group had significantly increased (P < 0.05) TNF-α levels compared to the NC group. In the same period, the T2 and T6 groups had significantly lower (P < 0.05) TNF-α levels than the PC group, and the T6 group showed significantly lower (P < 0.05) IL-6 levels compared to the PC group. At days 7 and 11 PI, the T6 group significantly decreased (P < 0.05) in both TNF-α and IL-6 compared to the PC group.

Table 7.

Effect of different ratios of phytogenic feed additives on serum concentrations of immunoglobulins and cytokines of weaned pigs challenged with Escherichia coli¹

Items	NC	PC	T1	T2	T3	T4	T5	T6	SE2	P-value
Day 0
IgG, mg/dL	237.00	216.67	216.00	210.67	232.00	221.67	218.33	213.67	25.033	0.995
IgA, mg/dL	1.00	1.00	1.00	1.00	1.33	1.33	1.00	1.00	0.105	0.061
TNF-α, pg/mL	112.65	100.24	120.54	134.68	127.75	105.09	104.83	130.24	15.966	0.691
IL-6, pg/mL	1478.78	1344.58	1543.14	1491.56	1525.30	1480.38	1430.27	1444.38	86.441	0.817
Day 2 PI
IgG, mg/dL	200.33	143.00	150.67	160.33	165.67	153.00	158.67	171.00	17.098	0.415
IgA, mg/dL	1.67	1.00	1.00	1.33	1.00	1.33	1.00	1.33	0.197	0.169
TNF-α, pg/mL	82.15b	112.05a	86.52b	81.34b	78.81b	97.57ab	82.67b	78.86b	4.983	<0.001
IL-6, pg/mL	1303.09ab	1569.85a	1385.74ab	1295.04ab	1234.92b	1430.53ab	1328.20ab	1180.45b	71.997	0.017
Day 4 PI
IgG, mg/dL	218.33	174.67	213.67	216.00	214.33	207.67	216.67	208.00	13.047	0.327
IgA, mg/dL	1.67	1.00	1.00	1.00	1.00	1.00	1.33	1.00	0.167	0.053
TNF-α, pg/mL	75.15bc	103.80a	84.49abc	72.39bc	73.95bc	89.74ab	70.95bc	68.20c	4.669	<0.001
IL-6, pg/mL	1233.65bc	1474.67a	1348.99abc	1283.82abc	1240.84bc	1389.58ab	1274.28abc	1144.74c	51.438	0.002
Day 7 PI
IgG, mg/dL	200.00	197.00	224.00	205.00	231.33	196.00	219.00	224.33	15.229	0.547
IgA, mg/dL	1.33	1.00	1.00	1.00	1.00	1.33	1.00	1.00	0.105	0.061
TNF-α, pg/mL	67.54ab	86.32a	78.84ab	72.17ab	76.59ab	80.78a	76.44ab	60.35b	4.384	0.006
IL-6, pg/mL	1018.76c	1367.53a	1235.32abc	1163.15abc	1274.19ab	1334.01a	1209.01abc	1094.18bc	50.286	<0.001
Day 11 PI
IgG, mg/dL	219.33	200.67	219.67	218.33	220.33	220.67	237.67	254.33	12.665	0.177
IgA, mg/dL	1.00b	1.00b	1.00b	1.00b	1.33a	1.00b	1.00b	1.00b	0.075	0.031
TNF-α, pg/mL	62.37bc	80.90a	74.67ab	69.84abc	73.87abc	61.16bc	63.43bc	60.77c	2.997	<0.001
IL-6, pg/mL	1001.10c	1259.43a	1160.77abc	1068.15bc	1209.32ab	1156.59abc	1107.66abc	1021.85c	39.282	<0.001

¹Abbreviation: NC, basal diet without E. coli challenge (negative control); PC, basal diet with E. coli challenge (positive control); T1, PC + PFA1 0.1%; T2, PC + PFA2 0.1%; T3, PC + PFA3 0.1%; T4, PC + PFA4 0.1%; T5, PC + PFA5 0.1%; T6, PC + PFA6 0.1%; IgG, immunoglobulin G; IgA, immunoglobulin A; TNF-α, tumor necrosis factor α; IL-6, interleukin-6; PI, post-inoculation; SE, standard error.

² n = 6 per treatment.

^a-^cMeans with different letters are significantly differ (P < 0.05).

Intestinal morphology

The T6 group had significantly improved (P < 0.05) VH compared to the PC group (Table 8; Fig. 2). The VH:CD had ­significantly increased (P < 0.05) in the T6 group compared to the T1 and T3 groups. The number of goblet cells in the villi had significantly higher (P < 0.05) in the PC group than in the NC group. The number of goblet cells in the crypt had significantly lower (P < 0.05) in the T3 group than in the T5 group.

Table 8.

Effect of different ratios of phytogenic feed additives on intestinal morphology of weaned pigs challenged with Escherichia coli¹

Items	NC	PC	T1	T2	T3	T4	T5	T6	SE2	P-value
VH, μm	374.72ab	331.01b	335.08b	389.12ab	388.33ab	372.06ab	360.78ab	406.55a	15.303	0.013
CD, μm	153.01	170.79	188.25	170.61	208.80	159.70	160.06	162.43	12.446	0.057
VH:CD	2.53a	2.01ab	1.88b	2.29ab	1.93b	2.33ab	2.28ab	2.52a	0.163	0.039
Villi goblet cells, n	17.83c	38.83a	33.83ab	33.50ab	21.17bc	28.17abc	35.33ab	28.33abc	4.675	0.039
Crypt goblet cells, n	20.67ab	31.83ab	29.17ab	26.67ab	20.33b	31.17ab	36.17a	23.67ab	3.444	0.022

¹Abbreviation: NC, basal diet without E. coli challenge (negative control); PC, basal diet with E. coli challenge (positive control); T1, PC + PFA1 0.1%; T2, PC + PFA2 0.1%; T3, PC + PFA3 0.1%; T4, PC + PFA4 0.1%; T5, PC + PFA5 0.1%; T6, PC + PFA6 0.1%; VH, villus height; CD, crypt depth; VH:CD, villus height to crypt depth ratio; SE, standard error.

² n = 6 per treatment.

^a-^cMeans with different letters are significantly differ (P < 0.05).

Figure 2.

Effect of different ratios of phytogenic feed additives on intestinal microscopic morphology (H&E staining) of weaned pigs challenged with Escherichia coli. NC, basal diet without E. coli challenge (negative control); PC, basal diet with E. coli challenge (positive control); T1, PC + PFA1 0.1%; T2, PC + PFA2 0.1%; T3, PC + PFA3 0.1%; T4, PC + PFA4 0.1%; T5, PC + PFA5 0.1%; T6, PC + PFA6 0.1%; Scale bar is 100 μm.

Relative protein expression of tight junction proteins

The PC group had significantly increased (P < 0.05) relative protein expression of calprotectin in the ileum compared to the NC group (Table 9). The T2 group significantly decreased (P < 0.05) relative protein expression of calprotectin compared to the PC group. The relative expression of CLDN1 had significantly lower (P < 0.05) in the PC group than in the NC group.

Table 9.

Effect of different ratios of phytogenic feed additives on relative protein expression of tight junction proteins of weaned pigs challenged with Escherichia coli¹

Items	NC	PC	T1	T2	T3	T4	T5	T6	SE2	P-value
Calprotectin, ng/mg protein	2.53c	5.36a	4.26abc	3.08bc	3.19abc	4.75ab	4.72abc	4.16abc	0.488	0.002
CLDN1, ng/mg protein	2.61a	1.27b	1.88ab	2.03ab	2.05ab	2.27ab	1.47b	1.75ab	0.228	0.005

¹Abbreviation: NC, basal diet without E. coli challenge (negative control); PC, basal diet with E. coli challenge (positive control); T1, PC + PFA1 0.1%; T2, PC + PFA2 0.1%; T3, PC + PFA3 0.1%; T4, PC + PFA4 0.1%; T5, PC + PFA5 0.1%; T6, PC + PFA6 0.1%; CLDN1, claudin 1; SE, standard error.

² n = 6 per treatment.

^a–cMeans with different letters are significantly differ (P < 0.05).

Discussion

During the postweaning period, weaned pigs are exposed to many stressors, such as dietary changes, separation from sows, the mixing of pigs from different pens, and adaptation to a new environment (Fairbrother et al., 2005). These factors induce significant changes in the gastrointestinal physiology, microbiology, and immunology of weaned pigs (Campbell et al., 2013; Heo et al., 2013). Harmful bacterial pathogens can invade through the damaged intestine, reducing the digestion and absorption of nutrients, and decreasing weaned pig’s growth performance (Gao et al., 2013; Song et al., 2022). In this study, an E. coli challenge resulted in decreased growth performance, such as reduced BW and ADFI. Diarrhea scores and serum pro-inflammatory cytokine levels had increased, and the expression level of CLDN1 had decreased due to the occurrence of intestinal inflammation. These symptoms are consistent with those observed in weaned pigs infected with E. coli in previous studies (Wang et al., 2020; Hong et al., 2021). Therefore, these results indicate that an oral challenge with pathogenic E. coli successfully induced a PWD model.

The ultimate purpose of our study was to investigate the effect of supplementing weaned pigs challenged with pathogenic E. coli with different ratios of PFA and determine the optimal mixing ratio for PWD prevention. In this study, PFA2 and PFA6 increased feed efficiency and decreased diarrhea over the entire period. Also, the PFA6 decreased serum levels of TNF-α and IL-6 and increased the VH:CD ratio. The PFA2 decreased the relative protein expression of calprotectin in the intestine. These findings suggest that supplementation with PFA2 or PFA6 could alleviate the negative effects of PWD, such as decreased growth performance and inflammation. However, to our knowledge, no previous studies have evaluated the effect of mixing different ratios of PFA in the diet of weaned pigs challenged with E. coli. Several studies fed a single PFA in pigs. Naringin and neohesperidin contained in BCE are the most abundant flavonoids in citrus fruits, with reported antioxidant and anti-inflammatory properties (Benavente-Garcia and Castillo, 2008). In weaned pigs, 50 mg/kg of pure naringin in the diet improved body weight gain and the feed conversion ratio (Goodarzi Boroojeni et al., 2018). According to Artuso-Ponte et al. (2015), the antibacterial and anti-inflammatory properties of naringin may improve the immune response and gut health of pigs, which could be a potential reason for improved growth performance. Cui et al. (2020) reported that supplementation with citrus extract increased the activity of alkaline phosphatase, lipase, and trypsin enzymes in the duodenum and jejunum of weaned pigs and might be involved in improving digestion and the absorption of lipids and protein in weaned pigs. Also, the citrus extract increased the VH:CD ratio in the duodenum and ileum (Sharma et al., 2016; Cui et al., 2020). This appears to improve intestinal morphology, as the intestine is repaired and protected through flavonoids’ anti-inflammatory and antioxidant effects in a citrus extract (Cui et al., 2020). Increases in the VH:CD ratio and GE digestibility of PFA6 in this study are also considered to be a positive effect of the flavonoids contained in BCE. According to an in vitro study using murine macrophages, the addition of naringin and neohesperidin in lipopolysaccharide (LPS)-treated cells decreased TNF-α levels, a pro-inflammatory cytokine increased by LPS challenge (Nakajima et al., 2017). In our study, PFA3 with 10% BCE decreased the levels of TNF-α up to day 4 PI, and PFA6 reduced TNF-α levels over the entire period. These results are consistent with previous studies in which PFA6 reduced the levels of pro-inflammatory cytokines such as TNF-α by protecting cells and tissues from inflammation-induced damage caused by oxidative stress via the antioxidant action of naringin and neohesperidin in BCE (Wu et al., 2003).

Grape by-products, such as grape seeds and grape marc included in PGE, contain large amounts of bioactive compounds that prevent oxidative stress and inflammation (Doshi et al., 2015; Taranu et al., 2019). Gessner et al. (2013) reported that feed efficiency was improved in weaned pigs fed 1% grape seed and grape marc extract. Another study found that grape by-products did not affect weaned pig’s growth performance but inhibited the LT of enterotoxigenic E. coli in an in vitro study (Verhelst et al., 2014). Also, grape by-products increased the abundance of beneficial bacterial species, including Bifidobacterium and Lactobacillus spp. (Griffin et al., 2017). Accordingly, the results suggest that the positive regulation of the gut microbiota by PGE of PFA2 and PFA6 may have reduced diarrhea in weaned pigs. According to Gessner et al. (2013), grape by-products increased the VH:CD ratio in the small intestine, thus improving digestion and the absorption of nutrients in the intestine. Also, in this study, PFA6 containing 10% PGE increased the ileal VH:CD ratio and recovered it to that in the NC group not infected with E. coli, and the digestibility of GE was also improved. However, another study showed the opposite result, where the growth of villi in the jejunum was inhibited in weaned pigs fed grape by-products (Sehm et al., 2007). Therefore, since PGE has not shown consistent results for improving intestinal morphology and the nutrient digestibility of weaned pigs, additional mechanistic studies on the effect of PGE on pig intestines are needed.

Essential oils, including thymol and carvacrol, are natural bioactive compounds with antioxidant properties and are generally known to be beneficial to animal health and performance (Liu et al., 2018; Omonijo et al., 2018). Several previous studies reported that essential oils could improve pig growth performance and nutrient digestibility (Zeng et al., 2015; Oh et al., 2018; Xu et al., 2018; Tian and Piao, 2019). Essential oils caused positive changes in intestinal lumen metabolites and increased the absorption of nutrients by improving digestive enzymatic activity, ultimately improving feed efficiency and nutrient digestibility (Windisch et al., 2008; Grilli et al., 2010). Previous studies showed that thymol and carvacrol reduced fecal coliform counts, inhibited biofilm formation by pig fecal isolates, and increased Lactobacilli counts in the intestine (Zou et al., 2016; Oh et al., 2017; Wei et al., 2017). Jiang et al. (2015) reported that the antibacterial action of essential oils reduced the viscosity of the intestinal digestion contents, thereby reducing the pathogen load in the pig gastrointestinal tract. These results suggest that PFA2 and PFA6 containing MEO decreased diarrhea scores in this study, as in our previous study (Chang et al., 2022). In our study, PFA2 containing 10% MEO decreased the relative protein expression of calprotectin in the ileum. Calprotectin is an abundant neutrophilic protein released during inflammation (Xiao et al., 2014). Calprotectin is highly correlated with the degree of inflammation, suggesting that the decreased calprotectin in PFA2 in this study suppressed intestinal inflammation in weaned pigs challenged with E. coli. CLDN1, a tight junction protein, is very important in sealing the pericellular space between epithelial cells, preventing intestinal bacteria from passing through the epithelium (Suzuki, 2013). In our previous study, supplementation with MEO significantly downregulated the expression of CLDN1 in the ilea of weaned pigs (Chang et al., 2022). According to Zou et al. (2016), essential oils promoted intestinal barrier integrity by regulating the intestinal bacterial and immune status of pigs. However, in this study, no differences in the expression level of CLDN1 were seen in the PFA-added treatment groups except for in the presence or absence of an E. coli challenge. Unlike previous studies, the results may have been different due to the difference in the type of PFA and their mixing ratio in this study.

As mentioned earlier, no previous studies evaluated the effect of the mixing ratio of PFA. Therefore, it is difficult to understand the exact mechanism of the effect of the mixing ratios of PFA on weaned pigs, and additional mechanistic studies are needed.

Conclusion

Supplementing with different ratios of BCE, MEO, and PGE in E. coli-induced weaned pigs alleviated the negative effects of pathogenic E. coli. Among them, the addition of PFA2 and PFA6 showed the effect of improving growth performance (increasing feed efficiency) and preventing diarrhea. In particular, the addition of PFA6 showed the effect of reducing the level of pro-inflammatory cytokines in serum (TNF-α, IL-6) and improving the intestinal morphology compared to E. coli-induced weaned pigs. Calprotectin, which is increased during the occurrence of intestinal inflammation, was significantly lower than that of E. coli-induced weaned pigs when PFA2 was added, suggesting that intestinal inflammation was alleviated. In conclusion, improvements in growth performance, diarrhea reduction, and immunity enhancement are demonstrated when 10% BCE, 20% MEO, 10% PGE, and 60% excipient are mixed. Therefore, it is considered to be the optimal ratio showing the greatest synergistic effect when the BCE, MEO, PGE, and excipients are fed in a ratio of 1:2:1:6. However, as there are no previous studies on the mechanism of the PFA mixing ratio, additional studies on the mechanism according to the PFA mixing ratio is needed.

Glossary

Abbreviations

ADFI

average daily feed intake

ADG

average daily gain

ATTD

apparent total tract digestibility

BCE

bitter citrus extract

BW

body weight

CD

crypt depth

CLDN1

claudin 1

CP

crude protein

DM

dry matter

GE

gross energy

H&E

hematoxylin and eosin

IgA

immunoglobulin A

IgG

immunoglobulin G

IL-6

interleukin-6

LPS

lipopolysaccharide

LT

heat labile toxin

MEO

microencapsulated blend of thymol and carvacrol

NC

negative control

PC

positive control

PFA

phytogenic feed additives

PGE

premixture of grape seed and grape marc extract, green tea, and hops

PI

post-inoculation

PWD

post-weaning diarrhea

TNF-α

tumor necrosis factor α

VH

villus height

VH:CD

villus height:crypt depth

WBC

white blood cell

Conflict of Interest Statement

The authors declare no conflicts of interest associated with this study.