Application of hyodeoxycholic acid in reducing animal fat deposition

Abstract

The present invention relates to the field of animal breeding technology, and in particular to the use of porcine deoxycholic acid in reducing animal fat deposition. The present invention uses UCP1 genetically modified pigs with a low-fat phenotype to conduct targeted metabolic analysis in feces, and discovers and identifies a pig intestinal endogenous metabolite that can reduce fat deposition, porcine deoxycholic acid, whose chemical formula is C 24 H 40 O4. The present invention finds that the content of swine deoxycholic acid can indicate the fat deposition ability of pigs, and oral administration of swine deoxycholic acid to mice can significantly reduce the fat deposition of mice. The swine deoxycholic acid provided by the present invention has the effect of reducing fat deposition, and can be developed as a feed additive for reducing pig fat deposition in the future. At the same time, it can be applied to the research and development of drugs for reducing human obesity, providing a scientific basis for the treatment of human obesity and other diseases.

Description

Technical Field

[0001] The invention relates to the technical field of animal breeding, in particular to the application of hyodeoxycholic acid in reducing animal fat deposition. Background Art

[0002] Porcine deoxycholic acid, also known as hyodeoxycholic acid, is a natural secondary bile acid that is abundant in pig and mouse bile. Existing literature reports that hyodeoxycholic acid has multiple physiological effects such as lowering blood lipids, antispasmodic, and antibacterial, and it has an important regulatory effect on the lipid metabolism of animal bodies. Adding 0.05%-1.00% hyodeoxycholic acid to feed can reduce the plasma cholesterol content of mice and improve the formation of atherosclerosis.

[0003] Pigs are one of the animals that are most likely to deposit fat, especially existing pig breeds, which have a very strong ability to deposit fat. Fat deposition is one of the most important economic traits of pigs and a typical complex quantitative trait. It not only affects meat quality, but is also closely related to lean meat rate and feed conversion efficiency. Therefore, for many years, reducing fat deposition and increasing lean meat rate has been one of the main goals of pig breeding. However, the number of key genes that regulate lipid metabolism has been discovered is relatively small, which has become a bottleneck faced by precise molecular breeding of fat traits. Summary of the invention

[0004] In order to solve the technical problems existing in the prior art, the present invention provides the use of hyodeoxycholic acid in reducing animal fat deposition.

[0005] In a first aspect, the present invention provides a metabolite capable of reducing animal fat deposition, named hyodeoxycholic acid, which can be used to reduce animal fat deposition.

[0006] The present invention further provides the use of porcine deoxycholic acid as a marker for identifying the fat deposition ability of pigs.

[0007] The present invention further provides the use of hyodeoxycholic acid in preparing medicine for treating obesity.

[0008] Furthermore, the chemical formula of the hyodeoxycholic acid is C 24 H 40 O4, the structural formula is as follows:

[0009]

[0010] In a second aspect, the present invention provides a method for identifying the fat deposition ability of a pig, comprising: detecting the content of porcine deoxycholic acid in pig metabolites, wherein the higher the porcine deoxycholic acid content, the lower the fat deposition ability of the pig.

[0011] Furthermore, the lower fat deposition ability includes any one or more of the following:

[0012] i) Have lower back fat thickness;

[0013] ii) Have a lower fat percentage;

[0014] iii) Have a higher lean meat ratio.

[0015] Furthermore, the detection of the content of porcine deoxycholic acid in porcine metabolites comprises:

[0016] Detect the content of porcine deoxycholic acid in the blood, feces, jejunal contents, ileal contents, cecal contents, colon contents or rectal contents of pigs.

[0017] Furthermore, the pigs include: one or more of Landrace pigs, Large White pigs, Duroc pigs, Bama pigs, Rongchang pigs, Northeast Min pigs, Guangdong and Guangxi Xiaohua pigs, Neijiang pigs, Ningxiang pigs, Jinhua Liangtouwu pigs, Taihu pigs, Chenghua pigs, Tibetan pigs, Laiwu pigs or Duroc-Changda three-way hybrid commercial pigs.

[0018] In a third aspect, the present invention provides a method for reducing animal fat rate, comprising:

[0019] The animals are fed with porcine deoxycholic acid in an amount of 0.1 to 1.5% of the body weight of the animals.

[0020] Further, the pigs are fed deoxycholic acid by any one or more of the following methods:

[0021] i) adding to feed for feeding;

[0022] ii) dissolved in edible oil for feeding;

[0023] iii) Direct oral feeding.

[0024] The animals that hyodeoxycholic acid can be used to reduce fat deposition in the host include pigs or mice (mice and rats), but are not limited to pigs or mice, and can be used in cattle, sheep, chicken livestock and cats, dogs and pets.

[0025] Furthermore, the animal is a wild-type animal, that is, an animal that has not undergone gene editing.

[0026] The present invention has the following beneficial effects:

[0027] The present invention uses UCP1 genetically modified pigs with a low-fat phenotype to conduct targeted metabolic analysis in feces, and discovers and identifies a pig intestinal endogenous metabolite that can reduce fat deposition, swine deoxycholic acid. Swine deoxycholic acid can be used as a marker to indicate the fat deposition ability of pigs. At the same time, feeding swine deoxycholic acid can effectively reduce body fat rate, which is of great value for the future development of feed additives for reducing animal fat deposition and treating human obesity.

[0028] In addition, the present invention provides a method and dosage of using hyodeoxycholic acid to reduce fat deposition, and the method is simple and easy to operate. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0030] Figure 1 It is a schematic diagram of the correlation between the content of porcine deoxycholic acid and fat accumulation index in the feces of UCP1 Bama pigs provided in Example 1 of the present invention; wherein A is the molecular structural formula of porcine deoxycholic acid; B is a volcano plot of targeted metabolism (bile acid) in the feces of wild-type Bama pigs and Bama pigs that specifically express UCP1 in adipose tissue (hereinafter referred to as UCP1 pigs), and the screening criteria are change multiple (FC)>1.5 and P<0.05; C is the content of porcine deoxycholic acid in the feces of WT pigs and UCP1 pigs; D is the correlation analysis of the content of porcine deoxycholic acid in the feces of WT pigs and UCP1 pigs and pig back fat thickness and fat rate.

[0031] Figure 2 Schematic diagram of the correlation between the content of porcine deoxycholic acid in serum and feces and gonadal fat, subcutaneous fat and perirenal fat indexes of mice fed with conventional diet (CD) and high-fat diet (HFD) after gavage with UCP1 pig feces provided in Example 1 of the present invention; wherein A is a flowchart of the fecal microbiota transplantation experiment; B is a weight graph of mice transplanted with fecal microbiota; C is a comparison graph of the weight and fat of mice in the UCP1 pig feces gavage group (UCP1, treatment group) and the WT pig feces gavage group (Con, control group) after slaughter after 28 days of CD and HFD; D is a comparison graph of subcutaneous fat and gonadal fat of mice in the treatment group and the control group after slaughter after 28 days of CD and HFD; E is a comparison graph of the weight and fat of mice in the treatment group and the control group after CD and HFD Comparison of gonadal fat, subcutaneous fat and perirenal fat after 28 days; F is the comparison of fecal dasycholic acid content between the treated group and the control group under HFD conditions; G is the comparison of serum dasycholic acid content between the treated group and the control group under HFD conditions; H is the correlation between serum dasycholic acid content and body weight (P<0.01), fat weight (P<0.01), subcutaneous fat (P<0.01) and gonadal fat (P<0.05) under HFD conditions.

[0032] Figure 3It is a schematic diagram of the correlation between the porcine deoxycholic acid content in the feces of lean meat pigs (Du Changda three-way hybrid commercial pigs) and fat pigs (Rongchang pigs) and the thickness of pig backfat provided by Example 1 of the present invention; wherein A is the backfat thickness of lean meat pigs (Du Changda three-way hybrid commercial pigs) and fat pigs (Rongchang pigs); B is the porcine deoxycholic acid content in the feces of lean meat pigs and fat pigs; C is the correlation between the porcine deoxycholic acid content in the feces of lean meat pigs and fat pigs and the thickness of pig backfat.

[0033] Figure 4 It is a schematic diagram of fat deposition in mice after oral administration of hyodeoxycholic acid provided in Example 2 of the present invention; wherein A is a weight curve of the wild type (WT group) and the oral hyodeoxycholic acid (HDCA group) at different time points; B is the slaughter index of the WT group and the HDCA group of mice after feeding with hyodeoxycholic acid for 28 days; C is a fat comparison picture of the WT group and the HDCA group of mice after feeding with hyodeoxycholic acid for 28 days; D is a fat content comparison picture of the WT group and the HDCA group of mice after feeding with hyodeoxycholic acid for 28 days. DETAILED DESCRIPTION

[0034] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the drawings of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0035] Unless otherwise specified, the technical means involved in the following embodiments can be carried out by conventional experimental means.

[0036] The experimental methods in the following examples are as follows:

[0037] The content of porcine deoxycholic acid in feces and serum was detected by multiple reaction monitoring technology (MRM) targeted assay;

[0038] The specific implementation method is as follows: firstly, the sample is extracted, then the sample is analyzed by chromatography-mass spectrometry, and finally the peak graph is extracted and corrected with a standard substance, so that the bile acid metabolites can be identified.

[0039] The three-point backfat average method was used to detect the thickness of pig backfat;

[0040] The specific implementation method is as follows: after slaughtering the pig, bleeding, removing the head, four hooves and tail, the remaining body is divided into two halves from the middle of the spine, and the thickness of the pig back fat at three points, namely the thickest part of the shoulder of the left half of the carcass, the junction of the thoracic and lumbar vertebrae, and the junction of the lumbar vertebrae, is measured with a vernier caliper, and the average value is calculated.

[0041] The fat percentage of pigs was tested using the slaughter cut method;

[0042] The specific implementation method is as follows: first, slaughter the pig, bleed it, remove the head, four hooves and tail, and then divide it into two halves along the middle of the spine. Separate the skin, meat, fat and bones from the right half of the carcass and weigh them separately. The fat weight obtained is divided by the sum of the skin, meat, fat and bone weights of the right half of the carcass to obtain the fat rate.

[0043] The gonadal fat, subcutaneous fat and perirenal fat of mice were tested by slaughter and weighing.

[0044] The specific implementation method is as follows: first weigh the mouse to obtain the mouse weight data, then slaughter the mouse, peel off the mouse gonadal fat, subcutaneous fat and perirenal fat respectively, and weigh them, so as to calculate the ratio of fat to body weight in each part.

[0045] Example 1

[0046] 1. The present invention identified porcine deoxycholic acid (such as Figure 1 The results of the study showed that the content of cholic acid in the feces of 6.5-month-old UCP1 Bama pigs and WT Bama pigs was significantly negatively correlated with fat rate, which may have the potential to reduce fat deposition. First, the fecal contents of 6.5-month-old UCP1 Bama pigs and WT Bama pigs were measured for targeted metabolism (bile acid). It was found that the content of cholic acid in the feces of UCP1 Bama pigs was significantly higher than that of Large White pigs. The correlation analysis of cholic acid in feces and slaughter phenotypes such as body weight, backfat thickness and fat rate was performed, and it was found that the content of cholic acid in feces was significantly negatively correlated with backfat thickness and fat rate (as shown in Figure 1). Figure 1 BD in ).

[0047] 2. The present invention further administered UCP1 pig and WT pig feces to antibiotic-treated mice, and fed them with conventional feed (CD) and high-fat feed (HFD). At the same time, the feces and serum of these mice were tested for the content of porcine deoxycholic acid, and the gonadal fat, subcutaneous fat and perirenal fat of these mice were tested. Figure 2The results shown in the figure, where A is the experimental flow chart of fecal microbiota transplantation; B is the weight of mice after fecal microbiota transplantation; C is the comparison of body weight and fat of mice in the UCP1 pig feces gavage group (UCP1, treatment group) and the WT pig feces gavage group (Con, control group) after slaughter after 28 days of CD and HFD; D is the comparison of subcutaneous fat and gonadal fat of mice in the treatment group and control group after slaughter after 28 days of CD and HFD; E is the comparison of gonadal fat, subcutaneous fat and perirenal fat of mice in the treatment group and control group after 28 days of CD and HFD; F is the comparison of porcine deoxycholic acid content in feces of mice in the treatment group and control group under HFD conditions; G is the comparison of porcine deoxycholic acid content in serum of mice in the treatment group and control group under HFD conditions; H is the correlation between porcine deoxycholic acid content in serum and body weight (P<0.01), fat weight (P<0.01), subcutaneous fat (P<0.01) and gonadal fat (P<0.05) under HFD conditions.

[0048] The results showed that the content of swine deoxycholic acid in the feces and serum of mice in the UCP1 pig feces gavage group (UCP1, treatment group) was significantly higher than that in the WT pig feces gavage group (Con, control group), and the content of swine deoxycholic acid in serum was significantly negatively correlated with fat.

[0049] 3. Finally, the present invention detects the content of porcine deoxycholic acid in the feces of fat pigs (Rongchang pigs) and lean pigs (Duchang-Changda three-way hybrid commercial pigs), and obtains the following Figure 3 The results shown in the figure showed that the content of porcine deoxycholic acid in the feces of lean pigs was significantly higher than that of fat pigs, and its content was significantly negatively correlated with back fat thickness.

[0050] The above results show that porcine deoxycholic acid is significantly negatively correlated with fat rate and has the potential to reduce fat deposition.

[0051] Example 2

[0052] This example studies the effect of feeding mice with porcine deoxycholic acid on fat deposition, and the specific process is as follows:

[0053] 1. Prepare 0.1 g / ml hyodeoxycholic acid oleic acid solution

[0054] In order to ensure that each mouse ingests 1‰ of its body weight of hyodeoxycholic acid, this embodiment first prepares a hyodeoxycholic acid oleic acid solution with a concentration of 0.1 g / ml. Specifically, 1 g of hyodeoxycholic acid (purity of 98.6%, Epsilon, Z-028) is weighed and dissolved in 10 ml of oleic acid (Solarbio, 08291), and a hyodeoxycholic acid oleic acid solution with a concentration of 0.1 g / ml is prepared.

[0055] 2. Oral administration of hyodeoxycholic acid oleic acid solution to mice by gavage

[0056] Weigh each mouse. The weight of the mouse in this embodiment is 20 grams. The mouse is gavaged with swine deoxycholic acid according to an oral dose of 1‰ of the body weight, that is, 20 grams × 1‰ = 0.02 grams. Therefore, 0.2 ml of swine deoxycholic acid oleic acid solution with a concentration of 0.1 g / ml is taken for gavage. The mice gavaged with swine deoxycholic acid are used as the experimental group (HDCA). At the same time, the mice gavaged with the same amount of oleic acid solution are used as the control group (Con). The experimental group mice and the control group mice are fed with high-fat diet at the same time, and the cycle is 28 days.

[0057] 3. Phenotypic assay found that oral administration of hyodeoxycholic acid significantly reduced fat deposition in mice

[0058] In this example, the body weight of the experimental group mice and the control group mice was measured once every 7 days after continuous intragastric administration for 28 days. The results showed that the body weight of the experimental group mice was significantly lower than that of the control group ( Figure 4 A in the figure). After 28 days of oral administration, the mice in the experimental group were slaughtered. Compared with the control group, the fat weight of the mice in the experimental group was significantly lower, while the lean body weight had no significant difference ( Figure 4 Similarly, the subcutaneous fat and gonadal fat of the experimental group mice were also significantly smaller than those of the control group ( Figure 4 C in the figure). Further analysis revealed that the ratio of subcutaneous fat, gonadal fat, and perirenal fat weight to body weight in the experimental group of mice was also significantly lower than that in the control group of mice ( Figure 4 The above results indicate that oral administration of hyodeoxycholic acid can significantly reduce fat deposition in mice.

[0059] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. The use of porcine deoxycholic acid as a marker for identifying the fat deposition ability of pigs, characterized in that: The fat deposition capacity of the pig includes the back fat thickness and fat rate of the pig.

2. A method for identifying the fat deposition ability of pigs, characterized in that: include: Detect the content of porcine deoxycholic acid in pig metabolites. Pigs with higher porcine deoxycholic acid content have lower fat deposition ability. The lower fat deposition ability includes any one or more of the following: i) Have lower backfat thickness; ii) Have a lower fat percentage; iii) Have a higher lean meat percentage.

3. The method according to claim 2, characterized in that The method for detecting the content of porcine deoxycholic acid in porcine metabolites comprises: Detect the content of porcine deoxycholic acid in the blood, feces, jejunal contents, ileal contents, cecal contents, colon contents or rectal contents of pigs.

4. The method according to any one of claims 2 to 3, characterized in that: The pigs include: one or more of Landrace pigs, Large White pigs, Duroc pigs, Bama pigs, Rongchang pigs, Northeast Min pigs, Guangdong and Guangxi small flower pigs, Neijiang pigs, Ningxiang pigs, Jinhua two-headed black pigs, Taihu pigs, Chenghua pigs, Tibetan pigs, Laiwu pigs or Duroc-Changda three-way hybrid commercial pigs.

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