Application of borneol essential oil in relieving piglet diarrhea

Abstract

The invention belongs to the technical field of livestock breeding, and particularly relates to application of borneol essential oil in relieving piglet diarrhea. The invention focuses on the application of the borneol essential oil in the field of piglet breeding, analyzes the effects of the borneol essential oil in the antibacterial and antioxidant capacities of piglet intestinal tracts, and reveals that the borneol essential oil improves the stress response capacity of piglets by regulating the activity of antioxidant enzymes of the piglets. Research finds that the borneol essential oil can significantly improve the resistance of piglets to stimulus by adjusting the activity of antioxidant enzymes in the piglets so as to further improve the form of the intestinal tracts of the piglets, which is represented by increasing the height and density of jejunum and rectum villus of the piglets and finally significantly improving the diarrhea rate of the piglets. The invention provides a systematic and reliable scientific basis and theoretical basis for the application of the borneol essential oil to prevention and alleviation of piglet diarrhea by using a nutrition regulation and control means in live pig breeding.

Description

Technical Field

[0001] The invention belongs to the technical field of animal husbandry, and particularly relates to application of borneol essential oil in alleviating diarrhea in piglets. Background Art

[0002] In the pig farming industry, the weaning stage is considered the 2-4 week age period, a crucial period for piglet growth and development. However, piglets often experience stress during the weaning process, leading to decreased feed intake, diarrhea, slowed growth and development, and even death. Diarrhea in early-weaned piglets is a common problem, posing a serious threat to pig farming profitability. According to statistics, deaths caused by pathogenic diarrhea account for over 40% of all piglet deaths, with enterotoxigenic Escherichia coli (ETEC) being one of the most common pathogens. ETEC colonizes the small intestine through the fimbriae and adhesins on its surface. After colonization, it secretes large amounts of enterotoxins, which interfere with the metabolic function of small intestinal epithelial cells, causing diarrhea and ultimately leading to dehydration and death in piglets.

[0003] In traditional animal husbandry practices, antibiotics are often used to prevent and treat diarrhea. However, the long-term and widespread use of antibiotics can lead to the development of drug resistance in pathogens. Furthermore, overuse of antibiotics can easily lead to drug residues in livestock and poultry products, which accumulate in the human body and pose a serious threat to human health. To address this issue, it is necessary to reduce the use of antibiotics in animal husbandry and develop green and safe feed additives to promote the development of "antibiotic reduction and elimination" in animal husbandry. Therefore, there is an urgent need to develop green and safe feed additives that can replace antibiotics to address the nutritional needs and health issues that may arise during the growth and development of piglets.

[0004] With the trend toward antibiotic-free farming, the application of traditional Chinese medicine in animal husbandry is becoming increasingly widespread. Among these, plant essential oils (PEO) are gaining increasing attention as a new antibiotic alternative. Essential oils are plant secondary metabolites, small molecules with aromatic odors extracted from plant parts such as flowers, stems, leaves, and roots through specific processes (such as pressing, steam distillation, and organic solvent extraction). Due to their environmentally friendly, safe, and highly effective properties, plant essential oils are becoming an ideal new animal feed additive.

[0005] Borneol essential oil (BEO) is a kind of plant essential oil, mainly extracted from the branches and leaves of Cinnamomum camphora. As a rare new species of camphor tree discovered for the first time in China, volatile oil containing D-borneol can be extracted from different parts of Cinnamomum camphora. Currently, the industry mostly uses its branches and leaves as raw materials to extract BEO through steam distillation. The main component of BEO is D-borneol (C. 10 H 18 BEO is a Chinese herbal medicine that contains borneol, which is a kind of Chinese herbal medicine that contains borneol. It is a kind of Chinese herbal medicine that contains borneol. BEO is a kind of Chinese herbal medicine that contains borneol. It ... BEO is a kind of Chinese herbal medicine that contains borneol. BEO is a kind of Chinese herbal medicine that contains borneol. BEO is a kind of Chinese herbal medicine that contains borneol.

[0006] Traditional plant essential oils are mostly liquid at room temperature. When exposed to air, they are volatile, easily oxidized, and temperature-sensitive. Some also have a strong odor. These characteristics not only limit the use of plant essential oils in feed but also hinder their optimal performance in animals. With the increasing research on natural active substances, microencapsulation technology can isolate the encapsulated substances from the external environment and release them only under specific conditions, thereby greatly protecting the active substances. Therefore, if essential oils are encapsulated using microencapsulation technology, the utilization rate of the active ingredients in the essential oils can be maximized, thereby improving their stability in additive applications. Therefore, microencapsulation technology provides a broader application scenario for the development and utilization of borneol essential oil. Summary of the Invention

[0007] In order to overcome the shortcomings of the above-mentioned prior art, the present invention uses ETEC K88 to challenge weaned piglets and explores whether microcapsule-encapsulated borneol essential oil has the effect of alleviating diarrhea and improving intestinal health in weaned piglets. This provides a scientific basis for the application of borneol essential oil in alleviating diarrhea in weaned piglets and also provides a theoretical reference for the high-value-added development and utilization of borneol essential oil.

[0008] In order to achieve the above object, the technical solution adopted by the present invention is:

[0009] The invention provides application of borneol essential oil in preparing a product for alleviating piglet diarrhea.

[0010] Preferably, the products include breeding feed and health-care medicines.

[0011] Preferably, the effective dose of borneol essential oil is 300-500 mg / kg.

[0012] More preferably, the effective dose of borneol essential oil is 400 mg / kg.

[0013] Studies have shown that adding borneol essential oil to the diet can regulate the activity of antioxidant enzymes in piglet serum to improve the piglet's antioxidant capacity, improve the piglet's body's redox state, and then improve the piglet's intestinal morphology, which is manifested by increasing the height and density of the piglet's jejunum and ileum villi, and ultimately significantly improving the piglet's diarrhea rate.

[0014] The present invention also provides a feed additive for alleviating piglet diarrhea, wherein the feed additive contains borneol essential oil as a main active ingredient.

[0015] Preferably, the feed additive comprises a microcapsule carrier and borneol essential oil on the carrier.

[0016] Microcapsule carriers mainly include carbohydrates, fats, proteins, gums, and cellulose derivatives. Among them, carbohydrate wall materials mainly include maltodextrin, corn syrup, beta-cyclodextrin, and modified starch; protein wall materials mainly include soy protein isolate, zein, whey protein, and gelatin; fat wall materials mainly include hydrogenated vegetable oil, stearic acid, palm oil, and lecithin; gums mainly include alginate, gum arabic, and xanthan gum; and cellulose derivatives mainly include methyl cellulose, ethyl cellulose, and cellulose acetate butyrate.

[0017] More preferably, the preparation method of the feed additive is: using palm oil as a carrier, mixing it with borneol essential oil, and allowing the essential oil to be adsorbed on the carrier.

[0018] Preferably, the feed additive is in the form of solid particles.

[0019] More preferably, the size of the solid particles is 20-30 mesh.

[0020] Preferably, the feed additive is used by adding it to the basic diet of piglets, mixing it well and then feeding it.

[0021] More preferably, the piglet basal diet consists of corn, soybean meal, puffed soybeans, fish meal, wheat bran, soybean oil, calcium hydrogen phosphate, stone powder, salt, 98% lysine, methionine, tryptophan, threonine, citric acid, and a premix, and the premix mainly comprises zinc monohydrate, multivitamin 436, antioxidant, choline, enzyme preparation, phytase, flavoring, sweetener, mildew inhibitor, compound mineral, and glucose oxidase.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The present invention focuses on the application of borneol essential oil in the field of piglet breeding, and deeply explores its mechanism of action in terms of antibacterial and antioxidant capacity of piglet intestine. Studies have found that borneol essential oil can significantly improve the piglet's resistance to stress by regulating the activity of antioxidant enzymes in piglets, and then improve the morphology of piglet intestines, which is manifested as increasing the height and density of piglet jejunum and ileum villi, and ultimately significantly improving the diarrhea rate of piglets. This discovery not only reveals the key role of borneol essential oil in piglet physiological regulation, but also provides a solid scientific basis and theoretical basis for the use of nutritional regulation means to prevent and alleviate piglet diarrhea in the process of pig breeding, and helps promote the scientific application of borneol essential oil in piglet breeding, such as as a piglet feed additive. Therefore, the present invention provides strong support for promoting the practical application of borneol essential oil in the prevention and control of piglet diarrhea and realizing green and efficient breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a diagram of the experimental design for challenging weaned piglets with ETEC K88;

[0025] Figure 2 H&E staining of the intestines of weaned piglets challenged with borneol essential oil and ETEC K88. Note: CON I: blank control group; CON II: ETEC-infected group; 100 mg / kg BEO: ETEC + 100 mg / kg BEO group; 400 mg / kg BEO: ETEC + 400 mg / kg BEO group. BEO: borneol essential oil. Representative histological images and shape parameter histograms of the duodenum, jejunum, and ileum (magnification x20). V / C = villus height / crypt depth. DETAILED DESCRIPTION

[0026] The following is a further description of specific embodiments of the present invention. It should be noted that the description of these embodiments is intended to facilitate understanding of the present invention and does not constitute a limitation of the present invention. In addition, the technical features involved in the various embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.

[0027] The experimental methods in the following examples are conventional methods unless otherwise specified, and the experimental materials used in the following examples are commercially available unless otherwise specified.

[0028] Example: Study on the application of borneol essential oil in alleviating diarrhea in piglets

[0029] 1. Materials and Methods

[0030] 1.1 Experimental Subjects

[0031] 21-day-old "Du×Chang×Da" three-way hybrid weaned castrated piglet.

[0032] 1.2 Test Site

[0033] Liuyang City Liuan Agricultural Yige Breeding Farm.

[0034] 1.3. Preparation of ETEC K88 bacterial culture

[0035] ETEC K88 strain was grown in Luria-Bertani broth at 37°C with shaking at 200 rpm for 12-16 hours, after which the bacterial culture was collected by centrifugation at 4000 rpm for 5 minutes for later use. Bacterial counts were performed using the dilution plate method and the OD 625nm The absorbance values of the bacterial solution were measured at 0h, 4h, 8h, 12h, 16h, 20h, and 24h (SYNERGYH1, Bio Tek, USA).

[0036] 1.4 Microencapsulation Technology and Microencapsulation of Borneol Essential Oil

[0037] Microencapsulation technology, a new technology recognized by the world's highest organizations, is a key 21st-century research and development process with widespread application in the food industry. Microencapsulation utilizes physical and chemical methods to encapsulate solid, liquid, or gaseous substances within a film-forming polymer, ultimately forming stable, sealed or semipermeable microcapsules. This technology isolates the core material from the outside world, improving its stability and extending the storage life of volatile substances. It also controls the release of functional core materials, enabling more targeted and controlled release during application. Furthermore, microencapsulation can mask odors, reducing the impact of irritating odors on product quality. It can also alter the state of matter, converting liquid or gaseous substances into solids for easier storage and transportation.

[0038] Microcapsule preparation methods can be broadly categorized as physical, chemical, physicochemical, and other novel methods. The main steps include carrier adsorption, granulation, primary coating, and secondary coating. In this study, palm oil was selected as the carrier for microencapsulation of borneol essential oil. This carrier was mixed with borneol essential oil and palm oil (borneol essential oil:palm oil = 1:4, w / w), allowing the essential oil to adsorb into the pores or surface of the carrier. Subsequently, the mixture was dispersed in water at a solid-liquid ratio of 1 mg:15 mL to form a suspension. This suspension was then spray-dried into fine droplets, which were then rapidly evaporated using hot air. The resulting white powdery solid particles were 24 mesh in size.

[0039] 1.5 Experimental Design and Grouping

[0040] like Figure 1As shown, 32 healthy, 21-day-old weaned castrated piglets of a Duroc × Chang × Da hybrid crossbreed weighing 6.36 ± 0.41 kg were randomly divided into four treatment groups based on similar body weights, with eight replicates (pens) per treatment and one pig per replicate. The experimental groups included: CON I (basic diet), CON II (basic diet), borneol essential oil (low) (basic diet + 100 mg / kg borneol essential oil), and borneol essential oil (high) (basic diet + 400 mg / kg borneol essential oil). The basal diet was formulated according to the National Research Council (NRC) guidelines for swine nutrition requirements (2012). The basal diet formula is shown in Table 1.

[0041] The pre-feeding period was 3 days, the main feeding period was 21 days, and samples were collected after 3 days of gavage with ETEC K88. During the gavage period, the CONⅡ group, the borneol essential oil (low) group, and the borneol essential oil (high) group were gavaged with 10 mL of ETEC K88 every day. 9 CFU / mL ETEC K88, while the CONⅠ group of experimental piglets were gavage-fed with an equal amount of normal saline (deprived of food and water for 3 hours after the challenge, and then had free access to food and normal drinking water). After fasting for 12 hours, all experimental piglets were weighed on the morning of the 22nd day, and then blood was collected from the anterior vena cava and slaughtered for sampling.

[0042] Table 1 Basic diet formula for piglets

[0043]

[0044] Note: The main ingredients of the premix (purchased from Hunan Lifeng Biotechnology Co., Ltd.) are zinc monohydrate, multivitamin 436, antioxidants, choline, enzyme preparations, phytase, flavoring agents, sweeteners, mildew inhibitors, compound minerals, and glucose oxidase.

[0045] 1.6 Test Management

[0046] The experiment adopted a full-in, full-out feeding and management model. Drinkers and feed troughs were cleaned before the experiment began, and routine disinfection and immunization procedures were followed. The pig house temperature was controlled at 25±3°C, and the humidity was controlled at 60% to 70%. The piglets were housed in separate pens with slatted plastic floors (1.2×2.0m), adjustable stainless steel feed troughs, and an automatic ventilation system. The piglets had free access to water and food. During the experiment, the pig house was dewormed and the pigs were immunized according to routine pig farm management procedures. The piglets' feeding and health were observed daily.

[0047] 1.7 Growth performance

[0048] Growth performance was measured with each replicate as the unit. Piglets were weighed at the beginning and end of the formal experimental period to calculate the average daily gain (ADG) of the piglets. The feed intake was counted and the average feed intake (ADFI) and feed-to-gain ratio (F / G) of the piglets were calculated.

[0049] Average daily gain (ADG) = (weight at the end of the experiment - weight at the beginning of the experiment) / number of days of the experiment;

[0050] Average daily feed intake (ADFI) = total feed intake during the experimental period / (number of experimental days × number of piglets);

[0051] Feed to gain ratio (F:G) = average daily gain / average daily feed intake.

[0052] At the same time, the scoring rules in Table 2 were used to score diarrhea, and the diarrhea rate was calculated according to the following formula:

[0053] Diarrhea rate (%) = total number of piglets with diarrhea / (total number of piglets tested × number of test days) × 100%.

[0054] Table 2 Diarrhea scores of piglets

[0055]

[0056] 1.8. Determination of serum antioxidant indexes

[0057] After standing for 30 minutes, the serum was prepared by centrifugation at 3500 r / min for 10 minutes. The serum was aliquoted and stored at -20°C until testing.

[0058] Kits produced by Nanjing Jiancheng Bioengineering Institute were purchased and serum antioxidant indicators were measured according to the kit instructions: total antioxidant capacity (T-AOC), glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) activities, catalase (CAT) and malondialdehyde (MDA).

[0059] 1.9. Intestinal sectioning and morphometric analysis

[0060] On day 22 of the experiment, the animals were weighed and slaughtered. During slaughter, the abdominal cavity was opened, and the intestinal segments were quickly removed and separated. The cecum was ligated, wrapped in tin foil, and stored in an ice box for intestinal microbial analysis. Approximately 3 cm long segments of the duodenum, jejunum, and mid-ileum were fixed with fixative and used to prepare intestinal tissue sections.

[0061] Tissue samples were washed, dehydrated, cleared, waxed, and embedded into wax blocks. Paraffin sections were then prepared using a microtome. These sections were then expanded, stained, and mounted. Images were captured using a microscope, and villus length and crypt depth were measured using CaseViewer. The ratio of villus length to crypt depth was then calculated for each treatment.

[0062] 1.10 Data Statistics and Analysis

[0063] All the original data were preliminarily sorted and calculated using Excel 2021, and then the Shapiro-Wilk test of SPSS 22.0 software was used to test the normality of the data. The data conforming to the normal distribution were analyzed by one-way analysis of variance (one-way ANOVA), and Duncan was used for multiple comparisons. All data results were expressed as mean and standard error of the mean (SEM). A P value less than 0.05 was considered statistically significant, and 0.05 < P < 0.10 was considered a trend.

[0064] 2. Experimental results

[0065] 2.1 Effects of BEO on growth performance and diarrhea rate of ETEC K88-challenged weaned piglets

[0066] The statistically relevant data on the growth of piglets were sorted and analyzed, as shown in Table 3 for details.

[0067] As shown in Table 3, compared with CONⅠ, there were no significant changes in the body weight, ADFI, ADG, and feed-to-weight ratio of piglets after adding BEO to the diet. However, adding BEO to the diet significantly reduced the diarrhea rate of piglets (P < 0.05). After ETEC challenge, compared with CONⅡ, there were significant changes in the body weight, ADFI, and ADG of piglets after adding BEO to the diet, but the difference in feed-to-weight ratio was not significant. Adding 100 mg / kg BEO to the diet had no significant difference in diarrhea rate, while adding 400 mg / kg BEO showed a significant difference in diarrhea rate. The results showed that adding BEO to the diet could improve the feed intake, daily weight gain, feed-to-weight ratio, etc. of weaned piglets, and when adding 400 mg / kg BEO, it could significantly improve the proportion of diarrhea piglets.

[0068] Table 3 Effects of borneol essential oil on growth performance and diarrhea rate of ETEC K88-challenged weaned piglets

[0069]

[0070] Note: Different lowercase letters superscripted on the same row of data indicate significant differences (P < 0.05), the same applies to the following table.

[0071] Note: CONⅠ: blank control group; CONⅡ: ETEC infection group; 100 mg / kg BEO: ETEC + 100 mg / kg BEO group; 400 mg / kg BEO: ETEC + 400 mg / kg BEO group. BEO: borneol essential oil.

[0072] 2.2 Effects of BEO on intestinal morphology of ETEC K88-challenged weaned piglets

[0073] Intestinal morphology and microvillus structure are one of the important indicators for evaluating the intestinal health of piglets. The integrity of its structure reflects the piglet's ability to digest and absorb nutrients. The small intestinal mucosa is the main site for digestion and absorption. Among them, villus height, crypt depth and villus density are key indicators for evaluating intestinal morphology. Therefore, HE staining is used to observe the morphology of the small intestinal villi of piglets to judge the intestinal health of piglets. The results are as follows Figure 2 and as shown in Table 4.

[0074] Depend on Figure 2 As shown in Table 4, compared with CON II, dietary supplementation with 100 mg / kg and 400 mg / kg BEO significantly increased villus height in the duodenum, jejunum, and ileum (P < 0.05). Furthermore, villus height in the jejunum and ileum of piglets in the BEO-supplemented group increased linearly (P < 0.05), reaching the highest value in the 400 mg / kg BEO group. Compared with CON II, dietary supplementation with 100 mg / kg and 400 mg / kg BEO significantly decreased crypt depth in the duodenum, jejunum, and ileum of piglets (P < 0.05). Furthermore, crypt depth in the duodenum and ileum of piglets in the BEO-supplemented group decreased linearly. Compared with CONⅡ, dietary supplementation with 100mg / kg and 400mg / kg BEO significantly increased the V / C ratio of the duodenum, jejunum, and ileum of piglets (P<0.05). The V / C ratio of piglets in the BEO supplementation group increased linearly (P<0.05), reaching the highest value in the 400mg / kg BEO group. The results showed that BEO treatment increased the height and density of villi in the jejunum and ileum of piglets, indicating that the addition of BEO to the piglet diet improved the intestinal morphology of piglets to a certain extent.

[0075] Table 4 Effects of borneol essential oil on intestinal morphology of weaned piglets challenged with ETEC K88

[0076]

[0077] Note: Data in the same row marked with different lowercase letters indicate significant differences (P<0.05).

[0078] Note: CONⅠ: blank control group; CONⅡ: ETEC-infected group; 100 mg / kg BEO: ETEC + 100 mg / kg BEO group; 400 mg / kg BEO: ETEC + 400 mg / kg BEO group. BEO: borneol essential oil.

[0079] 2.3 Effects of BEO on serum antioxidant enzyme activities in weaned piglets challenged with ETEC K88

[0080] The redox homeostasis maintained by the oxidative and antioxidant systems is crucial for overall health. Once this balance is disrupted, the body produces excessive ROS, causing oxidative stress and potentially leading to various complications. Numerous studies have shown that T-SOD, T-AOC, GSH, and MDA are important indicators of the body's antioxidant capacity. To further explore the mechanism by which borneol essential oil alleviates diarrhea in piglets, we measured antioxidant enzyme-related indicators in weaned piglets. The results are shown in Table 5.

[0081] As shown in Table 5, ETEC challenge reduced serum T-AOC, T-SOD, GSH-Px activity, and CAT levels in piglets, while increasing MDA and iNOS levels. Compared with CON II, dietary BEO supplementation had a significant effect on piglets. BEO at 400 mg / kg significantly increased T-AOC, T-SOD, CAT, and GSH-px levels (P < 0.05). BEO at 100 mg / kg significantly decreased MDA and iNOS levels (P < 0.05). These results suggest that dietary BEO enhances antioxidant capacity and improves redox status in piglets by regulating the activity of antioxidant enzymes in their serum.

[0082] Table 5 Effects of borneol essential oil on serum antioxidant enzyme activities in weaned piglets challenged with ETEC K88

[0083]

[0084] Note: CONⅠ: blank control group; CONⅡ: ETEC infection group; 100 mg / kg BEO: ETEC + 100 mg / kg BEO group; 400 mg / kg BEO: ETEC + 400 mg / kg BEO group; BEO: borneol essential oil.

[0085] In summary, the present invention analyzes the role of borneol essential oil in the antibacterial and antioxidant capacity of piglet intestines and reveals that borneol essential oil improves piglet's ability to cope with stress by regulating the activity of piglet antioxidant enzymes. The present invention provides a systematic and reliable scientific basis and theoretical foundation for the use of borneol essential oil in pig farming to prevent and alleviate piglet diarrhea through nutritional regulation.

[0086] The embodiments of the present invention are described in detail above, but the present invention is not limited to the described embodiments. It is apparent to those skilled in the art that various changes, modifications, substitutions, and variations of these embodiments may be made without departing from the principles and spirit of the present invention, and the changes still fall within the scope of protection of the present invention.

Claims

1. Application of borneol essential oil in preparing products for alleviating piglet diarrhea.

2. The use according to claim 1, characterized in that The products include breeding feed and health-care medicines.

3. The use according to claim 1, characterized in that The effective dosage of the borneol essential oil is 300-500 mg / kg.

4. A feed additive for alleviating diarrhea in piglets, characterized in that: The feed additive takes borneol essential oil as a main active ingredient.

5. A feed additive for alleviating piglet diarrhea according to claim 4, characterized in that, The feed additive comprises a microcapsule carrier and borneol essential oil on the carrier.

6. A feed additive for alleviating piglet diarrhea according to claim 5, characterized in that: The preparation method of the feed additive comprises the following steps: using palm oil as a carrier, mixing the palm oil with borneol essential oil, and allowing the essential oil to be adsorbed on the carrier.

7. A feed additive for alleviating piglet diarrhea according to claim 5, characterized in that: The dosage form of the feed additive is solid particles.

8. A feed additive for alleviating piglet diarrhea according to claim 7, characterized in that: The size of the solid particles is 20-30 mesh.

9. A feed additive for alleviating piglet diarrhea according to claim 4, characterized in that: The feed additive is used in the following manner: adding the feed additive to the basic daily diet of piglets, mixing the feed additive evenly and then feeding the piglets.

10. The feed additive for alleviating piglet diarrhea according to claim 9, characterized in that: The piglet basal diet consists of corn, soybean meal, puffed soybeans, fish meal, wheat bran, soybean oil, calcium hydrogen phosphate, stone powder, salt, 98% lysine, methionine, tryptophan, threonine, citric acid, and a premix; the premix mainly comprises zinc monohydrate, multivitamin 436, antioxidant, choline, enzyme preparation, phytase, flavoring agent, sweetener, mildew inhibitor, compound mineral, and glucose oxidase.

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