Effects of Macleaya cordata Water Extract on the Growth Performance, Diarrhea Index, and Intestinal Health of Weaned Piglets Challenged with Escherichia coli

Yang Fan, Li Yuying, Tian Junquan, Su Wenxuan, Bao Xuetai, Yao Kang
(1.Institute of Subtropical Agriculture, Chinese Academy of Sciences, Key Laboratory of Animal Nutrition Physiology and Metabolism, Hunan Province, Hunan Provincial Research Center for Healthy Livestock and Poultry Farming, Changsha 410125, China; 2.University of Chinese Academy of Sciences, Beijing 100049, China.)

 

Abstract

This experiment aimed to investigate the effects of adding Macleaya cordata water extract to the diet on the growth performance, diarrhea index, and intestinal health of weaned piglets challenged with enterotoxigenic Escherichia coli (ETEC). A total of 30 healthy “Duroc × Landrace × Large White” weaned piglets (average body weight of 8.22 ± 0.98 kg) at 28 days of age were randomly divided into three groups, with 10 replicates per group and 1 piglet per replicate. The control and model groups were fed a basal diet, while the experimental group received a basal diet supplemented with 1 mL/kg of Macleaya cordata water extract. The pre-experimental period lasted 3 days, and the formal experiment lasted 18 days. On the 15th day of the experiment, the model group and the experimental group were orally administered 10 mL of ETEC K88 bacterial solution (2×10^9 CFU/mL), while the control group received the same dose of sterile LB broth. The results showed:

  1. Before the challenge (Days 1–14), the average daily gain (ADG) of weaned piglets in the experimental group was significantly higher (P < 0.05), and the feed-to-gain ratio (F/G) was significantly lower (P < 0.05) compared to the control group. After the challenge (Days 15–18) and throughout the entire experimental period (Days 1–18), no significant differences in growth performance were observed among the groups (P > 0.05).
  2. 24 hours after the challenge, the diarrhea index of the model group was significantly higher (P < 0.05) than that of the control group. The experimental group had a lower diarrhea index than the model group, but the difference was not significant (P > 0.05). 48 hours after the challenge, the diarrhea index of the model group was still significantly higher than that of the control group (P < 0.05), while the experimental group had a significantly lower diarrhea index compared to the model group (P < 0.05).
  3. The model group had a significantly deeper jejunal crypt depth and a lower villus-to-crypt ratio (V/C) compared to the control group (P < 0.05). The experimental group showed a significantly higher V/C ratio and a shallower crypt depth in the jejunum (P < 0.05) compared to the model group.
  4. The relative mRNA expression of intestinal pro-inflammatory factors such as IL-1β, IL-17, TNF-α, and IL-8 was significantly higher in the model group compared to the control group (P < 0.05). The experimental group showed significantly lower expression of these inflammatory markers compared to the model group (P < 0.05).

In conclusion, the addition of 1 mL/kg Macleaya cordata water extract to the diet improved the growth performance of weaned piglets, alleviated diarrhea, and mitigated the intestinal inflammation and structural damage caused by Escherichia coli infection.

Keywords: Macleaya cordata water extract, weaned piglets, Escherichia coli, growth performance, diarrhea, intestinal morphology, inflammation

Chinese Library Classification Number: S816.7
Document Identification Code: A
Article Number: 1006-267X (2023) 01-0157-11

Introduction

The weaning stage is crucial for piglets, and early weaning is one of the key measures to improve the efficiency of modern pig farming systems. However, early weaning can also impose physiological and psychological stress on piglets, resulting in symptoms such as anorexia, intestinal structural and functional damage, diarrhea, and low resistance. At the same time, due to the incomplete development of the digestive and immune systems, weaned piglets under stress are more susceptible to pathogenic bacteria, which can further damage the intestinal structure and function, leading to vomiting, diarrhea, and other symptoms, ultimately threatening piglet health and increasing mortality, causing significant economic losses.

Enterotoxigenic Escherichia coli (ETEC) is one of the main pathogenic factors causing diarrhea in piglets. It can be transmitted through food, water, and feces, posing a severe threat to piglet health. After entering the piglet’s intestines, E. coli multiplies and begins secreting adhesins, which help it colonize the intestinal epithelium, and toxins such as enterotoxins, edema toxins, endotoxins, and hemolysins, which destroy the integrity and function of the intestinal mucosa, leading to diarrhea, vomiting, inflammation, and edema in piglets.

Traditionally, antibiotics have been the primary method to prevent and treat diarrhea in piglets, but research has shown that prolonged antibiotic use can lead to bacterial resistance, drug residues, environmental pollution, and public safety concerns. Furthermore, regulations in China now prohibit feed manufacturers from producing commercial feed containing antibiotic growth promoters. Therefore, finding new, natural, and environmentally friendly alternatives to antibiotics is imperative.

Macleaya cordata is a perennial herbaceous plant from the Papaveraceae family, widely distributed in China and several countries in Europe and North America. It is rich in isoquinoline alkaloids, including sanguinarine, chelerythrine, protopine, coptisine, and other compounds. Among them, sanguinarine and chelerythrine are the main active components, which have been shown to promote growth, alleviate inflammation, regulate intestinal flora, enhance immunity, and exhibit antibacterial properties.

Recent studies have reported the antimicrobial and anti-inflammatory properties of sanguinarine and chelerythrine. For example, sanguinarine and chelerythrine have been found to inhibit the growth of pathogenic bacteria like Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, and Streptococcus agalactiae. Sanguinarine also mitigates inflammation by inhibiting the activation of NF-κB-related genes. Furthermore, research on the use of Macleaya cordata extracts in animal production has shown improved growth performance and feed efficiency in weaned piglets, broilers, and calves.

However, the effects of Macleaya cordata water extract on the growth performance, diarrhea, intestinal morphology, and inflammation in piglets infected with E. coli have not been reported. Therefore, this study aims to explore the impact of Macleaya cordata water extract on weaned piglets challenged with ETEC, providing a reference for the application of Macleaya cordata extract in managing diarrhea in weaned piglets.

1. Materials and Methods

1.1 Experimental Materials

The Macleaya cordata water extract used in this experiment was self-made from the leaves of Macleaya cordata through crushing, heating, decoction, and concentration. The measured content of sanguinarine was 43.25 μg/mL. The Escherichia coli (ETEC) K88 strain used in this experiment was obtained from the National Center for Strain Preservation (No. VIP(S) 24607), and was prepared by resuscitation and serial propagation to obtain the required concentration of E. coli for the experiment.

1.2 Experimental Design and Management

A total of 30 healthy “Duroc × Landrace × Large White” weaned piglets at 28 days of age (average body weight: 8.22 ± 0.98 kg) were randomly assigned to three groups, with 10 replicates per group and 1 piglet per replicate. The three groups were the control group, the model group, and the experimental group. The control and model groups were fed a basal diet, while the experimental group was fed a basal diet supplemented with 1 mL/kg of Macleaya cordata water extract. The basal diet was formulated according to the nutritional standards of NRC (2012), and the composition and nutritional levels of the diet are shown in Table 1. The pre-experimental period lasted for 3 days, and the formal experiment lasted for 18 days. On the 15th day of the experiment, each piglet in the model and experimental groups was orally administered 10 mL of ETEC K88 bacterial solution at a concentration of 2 × 10^9 CFU/mL, while the control group was orally administered an equal volume of sterile LB broth.

This experiment was conducted at the animal facility of the Institute of Subtropical Agriculture, Chinese Academy of Sciences. The experimental site was strictly controlled with closed management. The piglets were housed individually in cages, with free access to feed and water during the experimental period. Regular cleaning and disinfection of the pigpens were carried out.

1) The premix provided the following per kilogram of the basal diet:

VA 12,000 IU,VB1 2.5 mg,VB2 4 mg,VB6 7 mg,VB12 20 mg,VD3 2,000 IU,VE 30 IU,VK3 2.5 mg,Biotin 80.00 μg,Folic acid 0.7 mg,D-pantothenic acid 12.5 mg,Nicotinic acid 40 mg,Cu (as copper sulfate) 6 mg,Fe (as ferrous sulfate) 100 mg,Mn (as manganese sulfate) 20 mg,Zn (as zinc sulfate) 100 mg,I (as potassium iodide) 0.4 mg,Se (as sodium selenite) 0.30 mg.

2) Nutrient levels were calculated values.

 

1.3 Sample Collection

On the 18th day of the experiment, the piglets were fasted for 12 hours but allowed free access to water. Then, blood samples were collected from the anterior vena cava, after which the piglets were euthanized, and their intestines (jejunum and ileum) were dissected. The intestinal contents were washed with saline 1–2 times, and the cleaned segments (2–3 cm) were wrapped in aluminum foil and quickly frozen in liquid nitrogen for further analysis.

1.4 Measurement Indicators and Methods

1.4.1 Growth Performance

Daily feed intake was recorded for each piglet. The body weight of each piglet was measured on days 1, 14, and 18 of the experiment to calculate average daily feed intake (ADFI), average daily gain (ADG), and feed conversion ratio (F/G). The calculation formulas were as follows:

  • ADFI = total feed intake / number of experimental days;
  • ADG = (final body weight – initial body weight) / number of experimental days;
  • F/G = ADFI / ADG.
1.4.2 Diarrhea Index

Diarrhea was observed and recorded daily at fixed times. Diarrhea severity was scored using a 4-level scale:

  • Normal (solid feces): score 0;
  • Mild diarrhea (soft, semi-formed feces): score 1;
  • Moderate diarrhea (semi-liquid feces, high moisture content): score 2;
  • Severe diarrhea (liquid feces, water separated): score 3.

The diarrhea index was calculated as:
Diarrhea index = diarrhea score / total number of piglets.

1.4.3 Intestinal Morphology

Jejunum and ileum tissue samples were fixed in 4% formaldehyde solution, sectioned, and stained for histological examination. Villus height and crypt depth were measured, and the villus-to-crypt ratio (V/C) was calculated.

1.4.4 Intestinal Immune Function

Real-time quantitative PCR was used to measure the relative mRNA expression levels of immune-related genes in the jejunum and ileum, including interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-1β (IL-1β), interleukin-12β (IL-12β), interleukin-17 (IL-17), interleukin-18 (IL-18), and tumor necrosis factor-α (TNF-α). The specific method was based on Fu et al. [20]. The relative mRNA expression levels of the target genes were calculated using the 2−ΔΔCt method, and the primer sequences for the genes are shown in Table 2.

1.5 Statistical Analysis

The experimental data were first processed using Excel 2019, followed by one-way ANOVA using SPSS 26.0. Duncan’s multiple range test was used for multiple comparisons. The results are expressed as “mean ± standard error.” P < 0.05 was considered statistically significant, and 0.05 < P < 0.10 was considered a significant trend.

2. Results and Analysis

2.1 Effects of Macleaya cordata Water Extract on Growth Performance of Weaned Piglets

Table 3 shows that during days 1–14 (before the challenge), there were no significant differences in final body weight, average daily feed intake (ADFI), and average daily gain (ADG), as well as feed-to-gain ratio (F/G) between the control group and the model group (P > 0.05). However, compared to the control group, the experimental group showed a slight increase in final body weight and ADFI, although the differences were not significant (P > 0.05). The ADG of the experimental group was significantly increased by 33.33% (P < 0.05), and the F/G was significantly reduced by 27.24% (P < 0.05).

During days 15–18 (after the challenge), the final body weight, ADFI, and ADG of piglets in the model group were slightly lower than those in the control group, and the F/G was slightly higher, but these differences were not significant (P > 0.05). There were also no significant differences in final body weight, ADG, and F/G between the experimental group and the model group (P > 0.05). Throughout the entire experiment (days 1–18), the final body weight, ADFI, and ADG of piglets in the experimental group were not significantly different from those in the control group (P > 0.05), but there was a trend towards a reduction in F/G (P = 0.089).

 

2.2 Effects of Macleaya cordata Water Extract on the Diarrhea Index of Weaned Piglets

As shown in Table 4, during days 1–14 (before the challenge), there were no significant differences in the diarrhea index among the three groups (P > 0.05). At 24 hours after the challenge, the diarrhea index of the model group was significantly higher than that of the control group (P < 0.05), while the diarrhea index of the experimental group increased slightly but was not significantly different from the control group (P > 0.05). Compared to the model group, the diarrhea index of the experimental group was slightly lower, but the difference was not significant (P > 0.05).

At 48 hours after the challenge, the diarrhea index of the model group was still significantly higher than that of the control group (P < 0.05). However, compared to the model group, the diarrhea index of the experimental group was significantly lower (P < 0.05).

2.3 Effects of Macleaya cordata Water Extract on the Intestinal Morphology of Weaned Piglets

From Table 5, it can be seen that, compared to the control group, the jejunal crypt depth of weaned piglets in the model group increased significantly (P < 0.05), while the ileal crypt depth slightly increased but the difference was not significant (P > 0.05). The jejunal villus-to-crypt ratio (V/C) decreased significantly (P < 0.05), but the weight of the jejunum and ileum, as well as the villus height in the jejunum, did not show significant differences (P > 0.05). Compared to the model group, the experimental group had a significantly higher jejunal V/C ratio, ileal villus height, and ileal V/C ratio (P < 0.05), while the jejunal crypt depth was significantly lower (P < 0.05). However, the differences in jejunal weight, villus height, and crypt depth were not significant (P >0.05).

Additionally, as shown in Figure 1, after ETEC infection, the ileal villus height in the model group of weaned piglets decreased, and the jejunal crypt depth increased, with obvious lesions and tissue damage. In contrast, compared to the model group, the experimental group and control group had higher villus heights in both the jejunum and ileum, lower crypt depths, and no visible tissue damage or necrosis.

2.4 Effects of Macleaya cordata Water Extract on the Intestinal Immune Function of Weaned Piglets

As shown in Table 6, compared to the control group, the model group had significantly higher relative mRNA expression levels of IL-1β in the jejunum (P < 0.05), while the mRNA expression levels of IL-18, IL-8, and TNF-α in the jejunum were slightly elevated but not significantly different (P > 0.05). Compared to the control group, the experimental group showed slightly lower mRNA expression levels of IL-18, IL-8, and TNF-α in the jejunum, while the relative mRNA expression of IL-1β was slightly increased, but none of these differences were significant (P > 0.05). However, compared to the model group, the experimental group had significantly lower mRNA expression levels of IL-18, IL-8, and IL-1β in the jejunum (P < 0.05), while TNF-α expression was reduced but not significantly (P > 0.05).

As shown in Table 7, compared to the control group, the model group had significantly higher mRNA expression levels of IL-17, IL-1β, TNF-α, and IL-8 in the ileum (P < 0.05). In contrast, the experimental group showed reduced mRNA expression levels of IL-17, IL-1β, IL-8, and IL-12β, although these differences were not significant (P > 0.05). Compared to the model group, the experimental group had significantly lower mRNA expression levels of IL-17, IL-1β, TNF-α, and IL-8 in the ileum (P < 0.05), while the mRNA expression of IL-12β was slightly reduced but not significantly different (P > 0.05).

3. Discussion

3.1 Effects of Macleaya cordata Water Extract on the Growth Performance of Weaned Piglets

In the early stages of weaning, piglets experience various types of stress, including nutritional, psychological, physiological, and environmental stress, which can negatively affect their feeding habits and gastrointestinal development, leading to diarrhea and impaired digestion and absorption of nutrients. This results in reduced growth performance. In previous studies, Macleaya cordata preparations have demonstrated excellent growth-promoting effects. Wang Min et al. reported that adding 300 mg/kg of Macleaya cordata powder to the diet reduced the feed-to-gain ratio and improved the overall growth performance of piglets. Similarly, adding 50 mg/kg of Macleaya cordata powder to the diet increased the average daily feed intake by 3.96%, increased the average daily gain by 9.08%, and reduced the feed-to-gain ratio by 4.67%.

In this experiment, during days 1–14 (before the challenge), the addition of 1 mL/kg of Macleaya cordata water extract to the diet significantly improved the average daily gain of weaned piglets and significantly reduced the feed-to-gain ratio, which is consistent with previous studies. The Macleaya cordata extract is thought to stimulate intestinal cell differentiation, promote intestinal development, and improve nutrient absorption, which may explain its effects on the growth performance of weaned piglets.

After the challenge, the experimental group showed a trend of improved average daily gain and feed-to-gain ratio compared to the model group, suggesting that Macleaya cordata water extract can mitigate the negative effects of Escherichia coli infection on growth performance. The extract’s ability to inhibit E. coli and reduce diarrhea likely contributed to its growth-promoting effects. Studies have shown that Macleaya cordata extract can inhibit the formation of the bacterial Z-ring, thereby interfering with bacterial division and reducing bacterial reproduction. Additionally, the extract can disrupt the bacterial cell membrane, inactivating the bacteria and reducing toxin production, which helps protect the intestinal structure and improve growth performance.

In summary, adding 1 mL/kg of Macleaya cordata water extract to the diet can improve the growth performance of weaned piglets and alleviate the decline in growth performance caused by E. coli infection.

3.2 Effects of Macleaya cordata Water Extract on the Diarrhea Index of Weaned Piglets

After weaning, piglets often experience physiological or pathogenic diarrhea due to the incomplete development of their digestive and immune systems. Escherichia coli K88 is one of the main pathogens causing diarrhea in piglets. Diarrhea can significantly affect piglets’ growth performance, increase their mortality rate, and result in substantial economic losses. Therefore, reducing the incidence of diarrhea is crucial for ensuring the healthy growth of piglets.

Previous studies have demonstrated that Macleaya cordata extract can alleviate diarrhea. For example, a study by Xia Chaodu et al. showed that adding 0.1% Macleaya cordata extract to the diet significantly reduced diarrhea rates in weaned piglets. Similarly, Wang Min et al. found that adding 300 mg/kg of Macleaya cordata powder to the diet significantly reduced diarrhea rates in weaned piglets.

In this experiment, 24 hours after the challenge, the diarrhea index of the model group was significantly higher than that of the control group, indicating that the E. coli infection model was successfully established. At 48 hours post-challenge, the diarrhea index of the experimental group was significantly lower than that of the model group, suggesting that Macleaya cordata water extract alleviated the diarrhea caused by E. coli infection. The anti-diarrheal effect of Macleaya cordata extract may be related to its ability to inhibit pathogenic bacteria and regulate the abundance of beneficial gut flora. This indicates that the extract can inhibit E. coli and protect the intestinal structure, thereby reducing the intestinal damage and diarrhea caused by E. coli infection.

A study by Liu et al. also found that Macleaya cordata extract reduced plasma D-lactate and diamine oxidase levels in growing pigs on day 14, indicating that the extract improved intestinal mucosal development and strengthened the intestinal barrier, thereby reducing diarrhea. Additionally, E. coli infection can trigger intestinal inflammation, which contributes to diarrhea. Sanguinarine, a key component of Macleaya cordata extract, has been shown to alleviate intestinal inflammation by acting on epithelial cells, promoting intestinal health and reducing diarrhea.

In summary, adding 1 mL/kg of Macleaya cordata water extract to the diet can alleviate diarrhea caused by E. coli infection in weaned piglets, likely through its antibacterial properties, protection of the intestinal structure, and reduction of intestinal inflammation.

3.3 Effects of Macleaya cordata Water Extract on Intestinal Morphology of Weaned Piglets

The normal structure of the intestine is crucial for maintaining the health and growth of piglets. Weaning stress can damage the intestinal structure, leading to villus atrophy, increased crypt depth, and impaired digestive, absorptive, and barrier functions. In addition, E. coli infection further damages the intestinal structure. Intestinal villus height, crypt depth, and the villus-to-crypt ratio (V/C) are important indicators for evaluating intestinal absorptive function. During normal development, intestinal epithelial cells from the crypt migrate to the villus, where they differentiate and mature, enhancing absorptive capacity. However, E. coli infection disrupts this process.

Chen et al. reported that Macleaya cordata extract significantly reduced crypt depth and increased the V/C ratio in the jejunum of weaned piglets, indicating that the extract can improve intestinal morphology. In this experiment, after the E. coli challenge, compared to the model group, the experimental group showed improved intestinal villus height, reduced crypt depth, and increased V/C ratio in both the jejunum and ileum. These findings suggest that adding Macleaya cordata water extract to the diet can mitigate the intestinal structural damage caused by E. coli infection.

The improvement in intestinal morphology may be due to the extract’s ability to inhibit pathogenic bacteria, thereby reducing toxin production and alleviating damage to the intestinal structure. Previous studies have shown that Macleaya cordata extract can effectively inhibit harmful intestinal bacteria in broilers, reducing the production of toxic substances and alleviating intestinal damage. Furthermore, Macleaya cordata extract can promote the proliferation and differentiation of intestinal cells, facilitating the repair and regeneration of damaged intestinal structures. In this study, after inactivating E. coli, Macleaya cordata extract likely promoted the regeneration of intestinal cells, helping restore the damaged intestinal structure to normal levels comparable to those in the control group.

In conclusion, adding 1 mL/kg of Macleaya cordata water extract to the diet can improve the intestinal morphology of weaned piglets by alleviating the villus atrophy and crypt hyperplasia caused by E. coli infection.

3.4 Effects of Macleaya cordata Water Extract on Intestinal Immune Function of Weaned Piglets

Macleaya cordata extract has demonstrated anti-inflammatory properties, which may be attributed to its active components, sanguinarine and chelerythrine. Sanguinarine can inhibit NF-κB activation and reduce the levels of tumor necrosis factor-alpha (TNF-α) and nitric oxide in macrophages, thereby alleviating inflammation. Additionally, both sanguinarine and chelerythrine have been shown to significantly reduce the mRNA expression of monocyte chemoattractant protein-1 (MCP-1) and IL-6 in macrophages, with effects similar to those of the anti-inflammatory drug prednisone.

After E. coli infection, piglet intestines produce toxins that damage the intestinal mucosa and stimulate the synthesis and secretion of pro-inflammatory cytokines, such as IL-6, IL-1β, and TNF-α, disrupting intestinal immune balance and leading to inflammation. The expression levels of inflammatory cytokines reflect the body’s inflammatory status. While a moderate inflammatory response is necessary for combating pathogens, excessive inflammation can cause tissue damage.

Previous studies have demonstrated the anti-inflammatory effects of Macleaya cordata extract. For example, Gao Huan et al. found that sanguinarine significantly reduced the relative mRNA expression levels of IL-6 and IL-8 in the intestinal mucosal cells of piglets, thereby alleviating inflammation in the intestinal epithelium. Furthermore, Macleaya cordata extract has been shown to reduce intestinal inflammation in broilers by down-regulating the expression of pro-inflammatory cytokines, such as IL-1β. The extract has also been reported to reduce the expression of inflammatory cytokines like macrophage migration inhibitory factor (MIF), IL-8, IL-1β, interferon-gamma (IFN-γ), and TNF-α, while increasing the expression of the anti-inflammatory cytokine IL-10, thereby alleviating rotavirus-induced intestinal inflammation in mice.

In this experiment, E. coli infection significantly increased the mRNA expression levels of IL-18, IL-8, and IL-1β in the jejunum, as well as IL-17, IL-1β, TNF-α, and IL-8 in the ileum of piglets in the model group, indicating the presence of intestinal inflammation. However, the addition of Macleaya cordata water extract significantly reduced the mRNA expression levels of these pro-inflammatory cytokines in both the jejunum and ileum of piglets in the experimental group. These findings suggest that Macleaya cordata water extract can alleviate intestinal inflammation by reducing the expression of pro-inflammatory cytokines such as IL-18, IL-8, IL-1β, IL-17, and TNF-α.

The anti-inflammatory and antibacterial properties of Macleaya cordata extract are likely responsible for its ability to reduce intestinal inflammation. However, the specific mechanisms underlying its anti-inflammatory effects remain unclear and require further investigation.

In conclusion, adding 1 mL/kg of Macleaya cordata water extract to the diet can alleviate the intestinal inflammation caused by E. coli infection in weaned piglets.

4. Conclusion

Adding 1 mL/kg of Macleaya cordata water extract to the diet can improve the growth performance of weaned piglets and alleviate the negative effects of Escherichia coli infection, including diarrhea, decreased growth performance, reduced villus height, increased crypt depth, and intestinal inflammation.

 

Reference