Method for testing influence of magnesium potassium sulfate on growth of weaned piglets

Abstract

The invention discloses a method for testing the influence of magnesium potassium sulfate on the growth of weaned piglets, which comprises the steps of in-vitro simulated digestive tract test and in-vivo animal test, preparation of simulated stomach and intestine digestive juice, detection of the dissociation rate of magnesium potassium sulfate in the simulated digestive juice, selection of 216 21-day-old weaned piglets, and random division of the 216 21-day-old weaned piglets into 6 groups, and relates to the technical field of livestock breeding. According to the method for testing the influence of the magnesium potassium sulfate on the growth of the weaned piglets, the dissociation and absorption rules of potassium and magnesium ions in the magnesium potassium sulfate in duodenum, jejunum and ileum of the weaned piglets are defined through in-vitro tests, and a theoretical basis is provided for dose design of in-vivo tests.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of livestock breeding, in particular to a test method for the influence of magnesium potassium sulfate on the growth of weaned piglets. BACKGROUND

[0002] Weaned piglets are in a physiological stress period, and are prone to problems such as reduced feed intake, increased diarrhea rate, weakened antioxidant capacity and intestinal flora imbalance, which seriously affect their growth and development and breeding benefits. At present, the industry often adjusts feed additives to improve the physiological state of weaned piglets, but there is a lack of a set of systematic test methods covering the "in vitro absorption mechanism-in vivo comprehensive effect" for the application of magnesium potassium sulfate in the feed of weaned piglets. The specific influence of magnesium potassium sulfate on the growth performance, antioxidant capacity, intestinal ion transport and flora structure of weaned piglets cannot be accurately quantified, resulting in a lack of scientific basis for the application of magnesium potassium sulfate in piglet breeding.

[0003] To solve the above problems, the application designs a complete scheme including in vitro simulated digestive tract test and in vivo animal test, which can comprehensively analyze the mechanism of action of magnesium potassium sulfate on weaned piglets and provide test support for the reasonable addition of magnesium potassium sulfate in piglet feed. SUMMARY

[0004] To solve the above technical problems, the application is implemented by the following technical scheme: The test method for the influence of magnesium potassium sulfate on the growth of weaned piglets is composed of in vitro simulated digestive tract test and in vivo animal test, and the specific steps are as follows: In vitro simulated digestive tract test Step 1, preparation of simulated digestive juice According to the physiological characteristics of the digestive tract of weaned piglets, simulated stomach and intestinal digestive juice containing pepsin, trypsin and bile acid is prepared, and the pH value and temperature are controlled. The pH of the stomach digestive juice is 2.0-3.0, the pH of the intestinal digestive juice is 6.5-7.5, and the temperature is 37 DEG C + / - 0.5 DEG C, which simulates the in vivo digestion environment. Step 2, PMS dissociation rate detection The magnesium potassium sulfate sample is added to the simulated digestive juice, and is oscillated at a constant temperature of 37 DEG C in a water bath, and the rotation speed is set to 100-120 r / min. Samples are taken at the start, 15 min, 30 min, 60 min and 120 min, and the concentrations of potassium ions and magnesium ions in the samples are detected by atomic absorption spectrometry to calculate the dissociation rate. Step 3, intestinal absorption rate detection The Ussing chamber was used to select the duodenum, jejunum and ileum tissues (thickness 0.3-0.5mm) of weaned piglets as perfusion membranes. Simulated intestinal juice (containing PMS) was injected into the mucosa side of the perfusion system, and buffer was injected into the serosa side, maintaining a constant temperature of 37°C and oxygen supply (O2:CO2=95:5). The serosa side buffer was collected at the beginning, 30min, 60min, 90min, and 120min, and the potassium ion and magnesium ion concentrations were detected to calculate the absorption rate of different intestinal segments.

[0005] In vivo animal experiments; Step 1, grouping and feeding management of test animals; Select 216 healthy 21-day-old weaned piglets, pig breed is Du×Chang×Da, initial body weight is 7.53±0.02 kg, and randomly divide into 6 groups, and the 6 groups are: Control group (basic feed), 0.15% PMS group (basic feed + 0.15% magnesium potassium sulfate), 0.30% PMS group (basic feed + 0.30% magnesium potassium sulfate), 0.45% PMS group (basic feed + 0.45% magnesium potassium sulfate), 0.60% PMS group (basic feed + 0.60% magnesium potassium sulfate), and 0.75% PMS group (basic feed + 0.75% magnesium potassium sulfate); The test period is 42 days, and all piglets are raised in the same environment during the period, the temperature is 24-26℃, the humidity is 60%-65%, and the piglets are free to eat and drink water, and the daily feed intake and diarrhea are recorded; Step 2, index detection; Growth performance detection: the average daily feed intake (ADFI) of the test from the 1st to the 42nd day is calculated, and the diarrhea rate from the 29th to the 42nd day is calculated, and the diarrhea rate calculation formula is: Diarrhea rate = number of piglets with diarrhea / total number of piglets × 100%; Blood index detection: after the test, 1 piglet with average weight is randomly selected from each repetition, blood is collected from the anterior vena cava, and serum is separated; biochemical analyzer is used to detect serum biochemical indexes, colorimetric method is used to detect serum antioxidant related enzyme activity (such as glutathione peroxidase GSH-Px), and enzyme-linked immunoassay is used to detect serum adrenaline and norepinephrine content; Intestinal tissue index detection: after blood sampling, the piglets were slaughtered, and the jejunum and colon mucosa were collected; the mucosal GSH-Px activity was detected by colorimetry, the mucosal interleukin 1β (IL-1β) content was detected by enzyme-linked immunoassay, and the mRNA expression of mucosal NOD-like receptor pyrin domain-associated protein 3 (NLRP3), cysteine protease 1 (Caspase-1), double control domain potassium channel 5 (KCNK5), transient potential cation channel 6 (TRPM6), and aquaporin 8 (AQP8) was detected by real-time fluorescent quantitative PCR (qPCR); Intestinal flora detection: the colon contents were collected, total DNA was extracted, and 16S rRNA gene V4-V5 region sequencing (using the Illumina MiSeq platform) was performed; the flora alpha diversity (Shannon index, Observed species index), and the relative abundance at the phylum / family / genus level (such as the Firmicutes phylum, the Ruminococcus family, and the Prevotella genus) were analyzed, and the correlation between the flora and physiological indicators (such as GSH-Px and IL-1β) was analyzed by Spearman correlation analysis; Step 3, data statistical analysis; SPSS software was used for data processing, one-way analysis of variance (ANOVA) was used to compare the differences between groups, linear regression analysis was used to analyze the dose-effect relationship between the PMS addition amount and the indicators, and P<0.05 was used as the significant difference standard.

[0006] The application provides a test method for the influence of magnesium potassium sulfate on the growth of weaned piglets. (1) The test method for the influence of magnesium potassium sulfate on the growth of weaned piglets determines the dissociation and absorption rules of potassium and magnesium ions in magnesium potassium sulfate in the duodenum, jejunum and ileum of weaned piglets through in vitro tests, thereby providing a theoretical basis for dose design in in vivo tests.

[0007] (2) The test method for the influence of magnesium potassium sulfate on the growth of weaned piglets covers multiple-dimensional indicators such as growth performance (feed intake, diarrhea rate), antioxidant capacity, intestinal immunity, ion channel and flora structure through in vivo tests, thereby comprehensively evaluating the comprehensive influence of magnesium potassium sulfate on weaned piglets and avoiding the limitations of single-index evaluation.

[0008] (3) The test method for the influence of magnesium potassium sulfate on the growth of weaned piglets is scientifically rigorous (random grouping, reasonable repeated setting and standardized statistical methods), and has high repeatability, thereby directly providing data support for the best addition amount (such as 0.30%-0.75%) of magnesium potassium sulfate in the feed of weaned piglets, and promoting the industrialized application of magnesium potassium sulfate in livestock breeding. DETAILED DESCRIPTION

[0009] The technical solutions in the embodiments of the present application will be described clearly and completely below. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work belong to the scope of protection of the present application.

[0010] The present application provides a technical solution: I. Details of in vitro simulation of digestive tract test Simulation of digestive fluid formula: simulated gastric juice: add pepsin (1.0 g), hydrochloric acid (adjust pH to 2.5) in 100 mL of deionized water; simulated intestinal fluid: add trypsin (0.5 g), bile acid (0.2 g), sodium bicarbonate (adjust pH to 7.0) in 100 mL of deionized water.

[0011] Dissociation rate detection: take 0.5 g of magnesium sulfate potassium and add it to 50 mL of simulated intestinal fluid, oscillate at 37℃, 110 r / min, centrifuge (8000 r / min, 10 min) at each time point, take the supernatant and detect the potassium and magnesium ion concentration with atomic absorption spectrometer (PEAA800).

[0012] Ussing perfusion system parameters: perfusion tank volume 10 mL, membrane area 1.13 cm², perfusion rate 5 mL / min, continuously pass 95% O2+5% CO2 mixed gas, maintain stable transmembrane potential.

[0013] II. Details of in vivo animal test Basic feed formula: refer to NRC (2012) piglet nutritional requirements, basic nutritional level: crude protein 21.0%, digestible energy 3.4 MJ / kg, lysine 1.3%, calcium 0.85%, total phosphorus 0.65%.

[0014] Diarrhea judgment standard: the feces of piglets is paste or water sample, adheres to the anus, which is determined as diarrhea.

[0015] qPCR detection conditions: reaction system 20 μL (SYBRGreenMix 10 μL, upstream and downstream primers each 0.5 μL, cDNA 2 μL, ddH2O 7 μL), reaction program: 95℃ pre-denaturation 30s; 95℃ denaturation 5s, 60℃ annealing 30s, 40 cycles; melting curve analysis (95℃ 15s, 60℃ 1min, 95℃ 15s).

[0016] 16S rRNA sequencing procedure: DNA extraction (CTAB method) -> PCR amplification (primers 515F: 5'-GTGCCAGCMGCCGCGGTAA-3', 907R: 5'-CCGTCAATTCMTTTRAGTTT-3') -> library construction -> Illumina MiSeq sequencing -> data analysis (using QIIME2 software).

[0017] It should be noted that, in the present document, relational terms such as first and second and the like can be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Moreover, the terms "comprises", "comprising", or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but can include other elements not expressly listed or inherent to such process, method, article, or apparatus. An element proceeded by "comprises... a" does not, without more constraints, exclude the presence of additional identical elements in the process, method, article, or apparatus that comprises the element.

[0018] While embodiments of the application have been shown and described, it is to be understood that the embodiments described are merely exemplary and that changes in form and detail can be made without departing from the spirit and scope of the application. The scope of the application is defined by the appended claims and their equivalents.

Claims

1. A test method for the effect of magnesium potassium sulfate on the growth of weaned piglets, characterized by, The in vitro simulated digestive tract test and the in vivo animal test are included, and the specific steps are as follows: The in vitro simulated digestive tract test is as follows: The simulated gastric and intestinal digestive juice is prepared, the dissociation rate of magnesium sulfate and potassium in the simulated digestive juice is detected, the Yush perfusion system is used, the duodenum, jejunum and ileum tissues of weaned piglets are used as the perfusion membrane, and the absorption rates of potassium and magnesium ions in different intestinal segments are detected; The in vivo animal test is as follows: Step one, 216 weaned piglets of 21 days old are selected and randomly divided into 6 groups; Step two, the test period is 42 days, the average daily feed intake from the 1st to the 42nd day is counted, and the diarrhea rate from the 29th to the 42nd day is counted; Step three, after the test is completed, the serum biochemical indexes, antioxidant and stress hormone indexes are detected by blood sampling, the emptying / colon mucosa is collected after slaughtering to detect the antioxidant, immune and ion channel mRNA indexes, and the intestinal content is collected to analyze the flora structure by 16S rRNA sequencing; Step four, the data is analyzed by ANOVA and linear regression, and P<0.05 is considered to be significant difference.

2. The test method for the effect of magnesium potassium sulfate on the growth of weaned piglets according to claim 1, characterized in that: In the in vitro simulated digestive tract test, the pH of the simulated gastric juice is 2.0-3.0, and pepsin is contained; The pH of the simulated intestinal juice is 6.5-7.5, and trypsin and bile acid are contained, and the temperature is maintained at 37 DEG C + 0.5 DEG C.

3. The test method for the effect of magnesium sulfate potassium on the growth of weaned piglets according to claim 1, characterized in that: The three ion channel mRNA indexes in step three include double control domain potassium channel 5, transient potential cation channel 6 and water channel protein 8.

4. The test method for the effect of magnesium potassium sulfate on the growth of weaned piglets according to claim 1, characterized in that: The flora indexes analyzed by 16S rRNA sequencing in step three include alpha diversity and relative abundance of firmicutes, rumen bacteria family or prevotella.