CN103245747A - Method for predicating metabolizable energy level of pig diets by metabonomics technology - Google Patents
Method for predicating metabolizable energy level of pig diets by metabonomics technology Download PDFInfo
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Abstract
The invention discloses a method for predicating the metabolizable energy level of pig diets by a metabonomics technology. The method is characterized in that the relative content of one to seven of eight plasma metabolic markers of a pig to be tested is plugged in equations with one to seven unknown quantities established in the invention to obtain the predicted value of metabolizable energy of the diets of the pig to be tested, wherein the plasma metabolic markers include lysophosphatidylcholine (18:3/0:0), myristoyl lysophosphatidylcholine (14:0/0:0), glycerophosphorylethanolamine (18:0/0:0), lysophosphatidylcholine (20:4/20:4), proline, phosphatidylcholine (18:2/0:0), arginylphenylalanylarginine and glycerophosphorylethanolamine (18:1(9Z)/0:0), and the relative deviation between the predicted value obtained from the equation with seven unknown quantities and an actual value does not exceed +/-1 percent. The method has significant theoretical and practical significances for evaluating the nutritional status and optimizing the formula in time, meeting the nutritional requirements of pigs under physiological and pathological conditions in different environments and improving the production efficiency of pigs.
Description
Technical field
The present invention relates to a kind of method of using metabonomic technology prediction swine rations metabolic energy level.
Background technology
Energy is the important nutritional labeling of feed, and the nutritional need of animal or nutrition supply are all based on energy, and energy feed also occupies maximum ratio simultaneously in feed cost.In recent years, because raw materials such as corn in the application of aspects such as manufacture alcohol, cause energy feed the more and more expensive phenomenon of shortage, price to occur.Therefore, how reasonable use energy in feed had both satisfied piglet for the needs of energy, can avoid the waste of energy feed again, just seemed particularly important.
In at present intensive Swine Production, the technological process according to all-in and all-out realizes swinery normally turnover on production line usually, with the feed daily ration of identical metabolic energy level of a collection of pig.This production technology has been ignored the difference of the individual and subpopulation of pig, and different pig farm and environment are to the influence of pig metabolizable energy nutritional need.In Swine Production, individual difference and different pig farm and environment can make identical nutrition condition produce different metabolin pedigree and production performance; Equally, different nutrition supplies also can produce specific metabolite profile and production performance.That is to say same energy level for different swinerys, effective energy value may be different.Therefore, the pig of Different Individual or subpopulation, varying environment, different health status is different to the needs of daily ration metabolizable energy.If when the preparation daily ration, do not consider the variation of its effective energy value, can be variant between the actual energy value of daily ration and expectation can be worth, if being worth greater than expectation, reality can be worth, then can cause the waste of feed resource, even pollute environment; If actual can the value less than expectation can be worth, then can reduce or suppress the production performance of pig.
Animal nutrition research becomes more meticulous day by day, and accurately the requirement of definite nutrient is regulated and control growth of animal has become domestic and international research focus.In order to reduce feed cost, to save feed resource and minimizing and raise pigs industry to the negative effect of environment, it is vital adopting the accurate effective energy value of raw material in the swine rations process for preparation.Intracorporal method is measured the raw material effective energy value wastes time and energy and the cost height, realizes predicting that by measuring material nutrient component also there are shortcomings such as predictablity rate is low in its method that can be worth thereby set up the dynamic regression model.
Metabolism group is the science of quantitative test biosome endogenous metabolism product kind, quantity and Changing Pattern thereof, and it understands the situation of biosome and the result of variations that internal and external factor causes by measuring end product of metabolism.Because the pattern-recognition advantage on its high-throughout information science characteristic and the basis thereof, by the correlation research of human nutriology's family expenses between factors such as recipe, life style and metabolin spectral pattern, and attempt the recognition mode of the metabolism group new standard as people's assessment of nutritional status is realized individuality or subpopulation recipe recommendation.Because the singularity of research object can't be carried out artificial recipe operation, the evaluation of mark depends on the clinical nutrition investigation and analyzes, and makes progress very slow.
Up to now, also do not utilize the metabolism group data to set up the dynamic regression predictive equation of blood plasma metabolic markers and daily ration effective energy value (metabolizable energy, digestible energy and net energy etc.), accurately predict the report of piglet diet effective energy value.
Summary of the invention
An object of the present invention is to provide quantitative detection following 1)-6) in the product of arbitrary described compound group or method detect or the metabolizable energy value of auxiliary detection daily ration that pig is fed in application:
1) compound L LL;
2) compound L LL and compound MLC;
3) compound L LL, compound GP0 and compd A LL;
4) compound L LL, compound GP0, compd A LL and proline;
5) compound L LL, compound GP0, compd A LL, compound L PC, arginyl phenylalanyl arginine, compound GP1;
6) compound L LL, compound GP0, compd A LL, proline, compound L PC, arginyl phenylalanyl arginine, compound GP1;
The structural formula of described compound L LL is suc as formula shown in the I:
The structural formula of described compound L PC is suc as formula shown in the II:
The structural formula of described compound GP0 is suc as formula shown in the III:
The structural formula of described compd A LL is suc as formula shown in the IV:
The structural formula of described compound GP1 is suc as formula shown in the V:
The structural formula of described compound MLC is suc as formula shown in the VI:
The present invention also provides a kind of and detects or the method for the metabolizable energy value of auxiliary detection daily ration that pig is fed, and comprises following A)-F) at least one in the step:
A) with in the X1 substitution equation 1, obtain ME1; Described equation 1 is ME1=3252.2+79.8 X1;
B) with in X1 and the X2 substitution equation 2, obtain ME2; Described equation 2 is ME2=3252.0+75.1 X1-75.0 X2;
C) with in X1, X3 and the X4 substitution equation 3, obtain ME3; Described equation 3 is ME3=3251.6+79.5 X1 – 53.7 X3 – 24.0 X4;
D) with in X1, X3, X4 and the X5 substitution equation 4, obtain ME4; Described equation 4 is ME4=3251.8+84.4 X1-45.8 X3 – 34.0 X4-42.5 X5;
E) with in X1, X3, X4, X6, X7 and the X8 substitution equation 5, obtain ME5; Described equation 5 is ME5=3251.6+95.5 X1 – 38.0 X3 – 20.8 X4 – 56.6 X6+52.7 X7-49.9 X8;
F) with in X1, X3, X4, X5, X6, X7 and the X8 substitution equation 5, obtain ME6; Described equation 6 is ME6=3251.7+96.0 X1-35.4 X3 – 28.4 X4 – 30.1 X5-46.6 X6+42.9 X7-40.2 X8;
Described ME1, ME2, ME3, ME4, ME5 and ME6 are the metabolizable energy value of daily ration that described pig to be measured is fed, and unit is kcal/kg;
Described X1 is the relative content of compound L LL described in the stripped blood plasma of pig to be measured;
Described X2 is the relative content of compound MLC described in the stripped blood plasma of pig to be measured;
Described X3 is the relative content of compound GPO described in the stripped blood plasma of pig to be measured;
Described X4 is the relative content of compd A LL described in the stripped blood plasma of pig to be measured;
Described X5 is the relative content of proline described in the stripped blood plasma of pig to be measured;
Described X6 is the relative content of compound L PC described in the stripped blood plasma of pig to be measured;
Described X7 is the arginic relative content of arginyl phenylalanyl described in the stripped blood plasma of pig to be measured;
Described X8 is the relative content of compound GP1 described in the stripped blood plasma of pig to be measured;
Described relative content is the lg value of getting the chromatographic peak area of respective compound in the stripped blood plasma of described pig to be measured.
In said method, described chromatogram is liquid chromatography, and the testing conditions of described liquid chromatography is specific as follows: chromatographic column is the quick high separation chromatographic column of Agilent ZORBAX UHV (ultra-high voltage) C-18; Flowing, to be respectively A mutually be the aqueous solution of 0.1% formic acid mutually for volumn concentration, and B is the acetonitrile solution of 0.1% formic acid mutually for volumn concentration; Flow velocity is 0.3mL/min, and temperature is 40 ℃, and from the B phase gradient wash-out of 5%-95%, be 25min analysis time.
In said method, before carrying out described chromatogram, the processing that the stripped blood plasma of described pig to be measured is comprised the steps: get described blood plasma and add among the extract A by the volume ratio of 1:4 and extract, obtain supernatant A, the volume ratio of pressing 1:1 in described supernatant A adds extract B, obtain supernatant B, described supernatant B is carried out described detection;
Described extract A is that methyl alcohol and acetonitrile are by the mixed solution of the volume ratio of 1:1;
Described extract B is that methyl alcohol and water are by the mixed solution of the volume ratio of 4:1.
In said method, the kind of pig described in the embodiments of the invention be Du Luoke * length white * Da Bai ternary growth pig, the body weight of described pig before the described daily ration of feeding is 13.4 ± 2.1kg, and when predicting the metabolizable energy value of daily ration that described pig is fed, the cycle of feeding of described daily ration is 28 days.
The present invention protects above-mentioned arbitrary described method in the daily ration of preparation pig or the application in the feed.
Described metabolizable energy is the energy that can deduct fecal energy, urine energy and the alimentary canal combustible gas of pig with feed.The energy that feed produces in the time of can referring to feed perfect combustion and produce end-product water and carbon dioxide.
The present invention is by the analysis of blood plasma end product of metabolism, blood plasma metabolic markers and the Changing Pattern thereof related with the daily ration metabolizable energy have been found out, and according to the different metabolic of feeding can level diet effective metabolic energy level of metabolin mark prediction daily ration that piglet is fed in the weanling pig blood plasma, set up the dynamic regression predictive equation of blood plasma metabolic markers and effective energy value, wherein, use the relative deviation of daily ration metabolizable energy predicted value that equation with one unknown quantity provided by the present invention obtains and actual value within ± 2%, use the relative deviation of daily ration metabolizable energy predicted value that seven yuan of equations provided by the present invention obtain and actual value within ± 1%.The present invention satisfies the nutritional need of pig under varying environment, physiology and the pathological conditions for timely evaluation nutrition condition and optimization of C, improves pig production efficiency and has important theory and practice significance.
Description of drawings
Fig. 1 is the blood plasma metabolism group principal component analysis (PCA) shot chart of feeding the weanling pig that different metabolic can level diet.Wherein, to represent the daily ration metabolic energy level of feeding respectively be 3150,3200,3250,3300 and the processed group result of 3350kcal/kg to icon 1-5.
Fig. 2 is blood plasma metabolism group partial least square method discriminatory analysis (PLS-DA) shot chart of feeding the weanling pig that different metabolic can level diet.Wherein, to represent the daily ration metabolic energy level of feeding respectively be 3150,3200,3250,3300 and the processed group result of 3350kcal/kg to icon 1-5.
Embodiment
Employed experimental technique is conventional method if no special instructions among the following embodiment.
Used material, reagent etc. if no special instructions, all can obtain from commercial channels among the following embodiment.
1, experimental animal
Test select for use healthy Du Luoke * length white * totally 180 of Da Bai ternary growth pigs, male and female half and half, the age in days that on average weans is 28 days, formally begins after 1 week of transition to test.Testing initial body weight is 13.4 ± 2.1kg, with initial body weight, is divided into 5 processing according to the randomized block experiment design, and each handles 6 repetitions, and every circle is a repetition, and each repeats 6 pigs.Male and female is separately raised.
2, feeding and management
Adopt all-in and all-out feeding and management pattern, the pig house temperature is controlled at 24-27 ℃, and the illumination program is 12h illumination/12h dark.Each circle (1.5 * 3.0m
2) all be furnished with 1 nipple-shape water fountain and 2 feed bunks.Net bed formula is raised periodic cleaning ight soil.Powder is fed, free choice feeding and drinking-water.Carry out immune expelling parasite according to pig farm Routine Management program.
3, test daily ration
The experimental basis daily ration is corn-dregs of beans type, uses corn and the soybean oil of different proportion to adjust daily ration metabolizable energy content.Handle the daily ration metabolic energy level for 5 and be respectively 3150,3200,3250,3300 and 3350kcal/kg, but standard ileum digestible lysine and metabolizable energy ratio (SID Lys:ME) maintenance 3.78g/Mcal.Simultaneously adjust other several essential amino acids (methionine, threonine and tryptophane) level according to ideal protein model (swine rearing standard, 2004), itself and lysine ratio are consistent.Add 0.25% chrome green as indicator.Daily ration prescription and nutritional labeling calculated value see Table 1.
4, data acquisition
Experimental period is 28 days.On-test when finishing, adopt electronic platform scale weighing piglet whose body weight and feed weight respectively, calculate average daily gain, average daily ingestion amount and weightening finish material consumpting ratio (perhaps feed efficiency).After the off-test, the piglet taboo is raised 12h, from every circle, select 3 piglets at random, gather vena cava anterior blood, blood sample is collected in the liquaemin anti-freezing vacuum test tube, places and is transported to the laboratory in the ice chest, 4 ℃, the centrifugal 15min of 3000rpm, separated plasma obtain the plasma sample that exsomatizes, and packing is stored in-80 ℃ of refrigerators.
5, data statistics
(general linear models GLM) carries out statistical study to the test figure of participation blood sampling piglet according to the general linear model in the randomized block experiment design employing SAS V8.02 statistical software.Be the test site group with initial body weight, total model comprises daily ration effect, block effect and stochastic error, and is as follows:
Y
ij=μ+T
ij+R
ij+ξ
ij
(i=1,2…,a,j=1,2…,b)
Y wherein
IjBe observed value, μ is population mean, T
IjBe daily ration effect, R
IjBe block effect, ξ
IjBe stochastic error.A is for handling number (being 5 in this test), and b is block number (being 6 in this test).If it is remarkable that each handles differences, then test with the Tukey multiple ratio.The result represents that with average and the average mistake of least square method P＜0.05 is significant difference.Adopt linear model and conic model to analyze daily ration different metabolic energy level to the influence of piglet growth performance.
6, result
Duration of test swinery general health is good.Daily ration different metabolic energy level sees Table 2 to the influence of piglet growth performance.Be that the processed group of 3350kcal/kg is compared with the daily ration metabolic energy level of feeding, the daily ration metabolic energy level of feeding be 3150 and the piglet final body representation work of the processed group of 3300kcal/kg improve (P＜0.05), the daily ration metabolic energy level of feeding be 3150,3200 and the piglet average daily gain of the processed group of 3300kcal/kg significantly improve.Rising along with the daily ration metabolic energy level, the average daily ingestion amount of piglet presents the trend of reduction, the daily ration metabolic energy level of feeding is that the processed group of 3150kcal/kg is the highest, and the daily ration metabolic energy level of feeding is minimum (linearity, P＜0.05 of the processed group of 3350kcal/kg; Secondary, P＜0.05).The daily ration metabolic energy level is to weight gain of piglets material consumpting ratio and feed efficiency do not make significant difference (P〉0.05).
Table 2 daily ration different metabolic energy level is for the influence of piglet growth performance
Annotate:
1The average mistake.
2Processing refers to multiple ratio result, and linearity and secondary be the effect of grain different metabolic energy level in a few days.In delegation, the female different table differential of shoulder marking-up different significantly (P＜0.05).
7, brief summary
Weanling pig and since its daily ration from liquid pig breast carry out the transition to solid-state feed, change that group feeding is supported and factors such as psychology variations cause stress, add that the feed intake of weanling pig is on the low side, sometimes even the negative growth of body weight can occur, it is bigger mainly to be that body fat loses.Many influence weaned piglet after in the trophism factor of growth performance, the dietary digestibility of energy level is particularly important.Therefore the material composition of high digestibility and high-energy ratio be to suitably improve in the weanling pig daily ration, indigestibility and the low feedstuff ratio of energy reduced.The average daily ingestion amount of the metabolic energy level appreciable impact piglet of daily ration, piglet can be regulated feed intake according to the concentration of energy in the daily ration, keeps the constant of energy intake.But metabolizable energy and digestible lysine (first limiting amino acid of pig) keep constant ratio in this test, even high-energy group piglet feed intake descends, do not have influence on the actual intake of piglet amino acid and metabolizable energy, simultaneously, the piglet daily gain is compared also on the low side with other groups, thereby feed efficiency is not made significant difference.
Under this test condition, the suitable daily ration metabolizable energy requirement that the comprehensive average daily ingestion amount of piglet is big, average daily gain and the less daily ration metabolizable energy of weightening finish material consumpting ratio are piglet is 3300kcal/kg, the economic benefit maximum namely expended minimum feed and obtained maximum weightening finish this moment.
Test daily ration and the trophic level of table 1 piglet different metabolic energy
11% interpolation of daily ration is pressed in premix in daily ration, for every kilogram of daily ration provides: vitamin A, 12,000IU; Vitamin D
3, 2,000IU; Vitamin E, 24IU; Vitamin K
3, 2.0mg; Cobastab
1, 2.0mg; Cobastab
2, 6.0mg; Cobastab
6, 4.0mg; Cobastab
12, 24 μ g; Nicotinic acid, 30mg; Pantothenic acid, 20mg; Folic acid, 3.6mg; Biotin, 0.40mg; Choline, 0.40g; Iron, 96mg; Copper, 8.0mg; Zinc, 120mg; Manganese, 40mg; Iodine, 0.56mg; Selenium, 0.40mg; Phytase, 120mg.
1, the processing of sample
The stripped plasma sample of the piglet of embodiment 1 is taken out the back in thawing on ice from-80 ℃ of refrigerators.Get 100 μ L blood plasma in every part, add 400 μ L metabolin extracts (methyl alcohol mixes with the volume ratio of 1:1 with acetonitrile) respectively, vortex concussion 5min ,-20 ℃ were extracted down after 1 hour, centrifugal 10min under 4 ℃, 13000rpm.The careful supernatant 200 μ L that draw place in the new centrifuge tube, dry up with nitrogen under the normal temperature, and redissolve in the methanol solution of 200 μ L 80%.Behind the concussion 5s, the centrifuging and taking supernatant places in the sample introduction bottle again, carries out high performance liquid chromatography-level Four bar-flight time tandem mass spectrum (HPLC Q-TOF MS) and detects.
2, the condition of HPLC Q-TOF MS detection
Instrument: HPLC Q-TOF MS detection system (HPLC(1290 series, Agilent company) and Q-TOF MS(6520 series, Agilent company).
The condition of high performance liquid chromatography: chromatographic column is the quick high separation chromatographic column of Agilent ZORBAX UHV (ultra-high voltage) (C-18,3.0 * 100mm, 1.8 μ m), flowing, to be respectively A mutually be the aqueous solution of 0.1% formic acid mutually for volumn concentration, and B is the acetonitrile solution of 0.1% formic acid mutually for volumn concentration; Flow velocity 0.3mL/min, 40 ℃ of temperature, from the B phase gradient wash-out of 5%-95%, be 25min analysis time.
Mass spectral condition: ion gun is electron spray holotype ESI+, 350 ℃ of dry gas temperature, dry gas flow velocity 12L/min, capillary voltage 3500V, cracked voltage 150V, acquisition quality scope 60-1000m/z, acquisition rate 2spectra/s.
3, data are handled
Detect through the HPLC of step 2 Q-TOF MS, obtain the metabolism group profile raw data of each sample, (version B.03.01 to use MassHunter Qualitative Analysis software, Agilent company), utilize the characterization of molecules extraction algorithm that the metabolism group profile raw data of all samples is carried out the calibration of background deduction, spectrum peak accurate mass number and retention time, extract the characteristic of retention time, mass-to-charge ratio and the ionic strength value of each sample.Data importing Mass Profiler Professional(is called for short MPP, version number B.02.00, Agilent company), after the filtration and correction of peak match, alignment, default parameter, the compound that filters out is carried out normalized (being the lg conversion) again, obtain proofreading and correct the data set of back metabolin characterization of molecules.Carry out packet and screening, statistical study, the analysis of variation multiple again, obtain the biomarker of difference after, the result of false sun (the moon) property is got rid of in the inspection of again data being extracted chromatography of ions figure (EIC).
4, principal component analysis (PCA) (PCA)
Utilize data that MPP software obtains step 3 to seek linear relationship between original variable by principal component analysis (PCA), form new variable.These major components are arranged by the reserving degree size order to the raw data variance, each other quadrature.The PCA shot chart as shown in Figure 1.Fig. 1 result shows that the piglet plasma sample of the processed group of the different metabolic of feeding energy level diet obviously cluster arrives together, and first principal component and Second principal component, can be explained 44.64% and 24.27% group difference respectively.This explanation piglet that different metabolic can level diet of feeding, there is significant difference in the metabolite profile of its blood plasma.
5, the foundation of partial least squares regression model and prediction
Use the partial least square method discriminatory analysis (PLS-DA) in the MPP software, set up the multiparameter model sample is predicted.The metabolism of handling through the step 3 spectrum data of randomly drawing 5 samples from each processed group are carried out modeling as being used for setting up the sample (training set) that model investigates, selection has the variable of appreciable impact to sample classification, the metabolism of handling through the step 3 spectrum data of remaining sample are verified model as the checking sample (test set) of multiple cross validation screening model and predicted, finally obtain reliable and stable pattern recognition model.By five groups of blood plasma PLS-DA shot charts (Fig. 2) as seen, the blood plasma metabolism spectrum of the growth pig of the five kinds of different metabolic energy level diet of feeding can distinguish fully, accumulates in different locus respectively.Identification and the predictive ability of model are as shown in table 3, and the model of foundation has stronger recognition capability to grouping, do not have the grouping of wrong identification to occur.
Table 3 utilizes recognition capability and the predictive ability of model after the PLS-DA modeling
6, the evaluation of blood plasma difference metabolic markers
From step 5, grouping had and finally in the variable of appreciable impact selects existing big covariance coefficient, the variable that big related coefficient arranged again for characterize should " metabolic energy level model " mark.The compound exact mass number that obtains by HPLC Q-TOF MS mass spectrum result carries out the generation of molecular formula, simultaneously at METLIN, HMDB, KEGG, the compound structure that database retrievals such as LIPIDMAPS are possible, to qualified compound, the MS/MS that carries out under the different cracking energy analyzes, the second order ms figure and the candidate markers that obtain are carried out binding analysis, compare with part existing standard product second order ms simultaneously, finally identify following 10 kinds of compounds (table 4) relevant with the daily ration metabolic energy level: amino acid and polypeptide class: lysine (hereinafter to be referred as LYS), phenylalanine (hereinafter to be referred as PHE), proline (hereinafter to be referred as PRO) and arginyl phenylalanyl arginine (hereinafter to be referred as APA); Lipid: lysophosphatidyl choline (18:3/0:0) (hereinafter to be referred as ALL); Glycerophosphorylethanolamine (18:0/0:0) (hereinafter to be referred as GP0); Phosphine acyl choline (18:2/0:0) (hereinafter to be referred as LPC); Lysophosphatidyl choline (20:4/20:4) (hereinafter to be referred as ALL); Glycerophosphorylethanolamine (18:1 (9Z)/0:0) (hereinafter to be referred as GP1) and cool acyl group hemolytic phosphatid ylcholine (14:0/0:0) (hereinafter to be referred as MLC).
The feed qualification result of endogenous difference metabolic markers in the weanling pig blood plasma that different metabolic can level diet of table 4
Annotate: W
l, W
2And W
3Be respectively metabolic markers in major component 1,2, the weighted value in 3.
In the table 4, the structural formula of described compound L LL is suc as formula shown in the I:
The structural formula of described compound L PC is suc as formula shown in the II:
The structural formula of described compound GP0 is suc as formula shown in the III:
The structural formula of described compd A LL is suc as formula shown in the IV:
The structural formula of described compound GP1 is suc as formula shown in the V:
The structural formula of described MLC is suc as formula shown in the VI:
7, brief summary
The weanling pig of the different metabolic of searching for food energy level diet, there is significant difference in the metabolite profile in the blood plasma.Principal component analysis (PCA) and partial least square method discriminatory analysis result show: the piglet blood plasma of the five kinds of daily rations of searching for food obviously cluster arrives together, and blood plasma metabolism spectrum can distinguish fully.Discrimination model recognition capability and the predictive ability height set up can be with accurately differentiating different daily ration metabolic energy level.Simultaneously, identify and the closely-related 10 species diversity metabolic markers of daily ration metabolic energy level, be mainly amino acid peptide class, organic acid and lipoid substance.
The foundation of embodiment 3, weanling pig daily ration metabolic energy level predictive equation
The purpose of present embodiment is the analysis by the blood plasma end product of metabolism, find out the metabolic markers related with production performance and nutrition parameters and Changing Pattern thereof, inquire into and how to predict the effective metabolic energy level of piglet according to the metabolin mark in the different metabolic energy level diet weanling pig blood plasma of searching for food, set up the dynamic regression predictive equation of blood plasma metabolic markers and effective energy value, attempt only by one or more blood plasma metabolic markers, accurately predict the effective energy value of piglet, for timely evaluation nutrition condition and optimization of C, satisfy varying environment, the nutritional need of pig under physiology and the pathological conditions improves pig production efficiency and has important theory and practice significance.
1, the relative content of difference metabolic markers
To get lg value (namely carrying out the lg conversion) according to the chromatographic peak area of 10 kinds of blood plasma difference metabolic markers of sample in each processed group of step 3 acquisition among the embodiment 2, namely obtain the relative content data (result as shown in table 5) of each metabolic markers in each sample.Carry out normal distribution-test and variance analysis with JMP 9 statistical softwares, carry out the Tukey multiple ratio during significant difference.Various blood plasma metabolic markers are in 5 kinds of daily ration metabolic energy level condition allowance below nominal size heteropoles remarkable (P＜0.01).
2, the correlation analysis of difference metabolic markers and daily ration metabolizable energy value
The relative content The data JMP Multivariate of the statistical software method of each difference metabolic markers is carried out correlation analysis, and the result is as shown in table 6.The result shows, except glycerophosphorylethanolamine (18:0/0:0), proline and cool acyl group hemolytic phosphatid ylcholine (14:0/0:0) content and daily ration metabolic energy level negative correlation, the content of all the other 7 kinds of metabolins all with daily ration metabolic energy level positive correlation, especially lysophosphatidyl choline (18:3/0:0) and daily ration metabolic energy level correlativity very high (0.828); Blood plasma phosphine acyl choline (18:2/0:0) and arginyl phenylalanyl arginine-level and daily ration metabolic energy level also have nearly 60% correlativity.
The feed relative content of the weanling pig blood plasma difference metabolic markers that different metabolic can level diet of table 5
Annotate: the Tukey multiple ratio is carried out in P＜0.05, in delegation, and the female different table differential of shoulder marking-up different significantly (P＜0.05).
The relative content of table 6 difference metabolic markers and the related coefficient of daily ration metabolic energy level
Annotate: ME is the metabolizable energy value of daily ration.
3. the regretional analysis of blood plasma metabolic markers and daily ration metabolizable energy
Adopt the stepwise among the JMP 9 to carry out simple regression analysis and stepwise regression analysis showed (stepwise regression analysis), with the relation of research daily ration metabolic energy level and difference metabolic markers.After table 7 has been listed daily ration metabolic energy level and blood plasma difference metabolic markers has been carried out regretional analysis, the prediction regression equation of the metabolizable energy of foundation.The P value is unlisted greater than 0.05 equation.Lysophosphatidyl choline (18:3/0:0) can be used as the simple regression equation that unique predictor is set up the effective metabolic energy level of piglet diet, and equation is: ME1=3252.2+79.8X1, the coefficient of determination of its correction (Adj R
2) be 0.682, predictive ability is better.In addition, the foundation of multiple regression equation has improved the accuracy of predictive equation, sets up the metabolizable energy predictive equation by 2 species diversity metabolic markers to 7 kind of metabolic markers, makes Adj R
2Be increased to 0.863 from 0.784.
Binary to seven a yuan equation is respectively: equation 2:ME2=3252.0+75.1 X1 – 75.0 X2(Adj R
2=0.784); Equation 3:ME3=3251.6+79.5 X1 – 53.7 X3 – 24.0 X4(Adj R
2=0.834); Equation 4:ME4=3251.8+84.4 X1-45.8 X3 – 34.0 X4-42.5 X5(Adj R
2=0.852); Equation 5:ME5=3251.6+95.5 X1 – 38.0 X3 – 20.8 X4 – 56.6 X6+52.7 X7-49.9 X8(Adj R
2=0.856).Best predictive equation is 7 yuan of equations, namely sets up predictive equation by 7 kinds of blood plasma difference metabolins: ME6=3251.7+96.0 X1-35.4 X3 – 28.4 X4 – 30.1 X5-46.6 X6+42.9 X7-40.2 X8, (Adj R
2=0.863).
Described X1, X2, X3, X4, X5, X6X7 and X8 are respectively the relative content of compound (being metabolic markers) LLL, MLC, GP0, ALL, PRO, LPC, APA and GP1 described in the stripped blood plasma of pig.
4. brief summary
This test findings shows that blood plasma difference metabolic markers can finely must be predicted weanling pig daily ration metabolic energy level, and the monobasic predictive equation is ME=3252.2+79.8 X1(Adj R
2=0.682), the optimum prediction equation is ME=3251.7+96.0 X1-35.4 X3 – 28.4 X4 – 30.1 X5-46.6 X6+42.9 X7-40.2 X8, (Adj R
2=0.863).
The linear regression predictive equation of table 7 weanling pig daily ration metabolic energy level
Annotate: Adj R
2Coefficient of determination for revising is used for estimating the goodness of fit of regression equation.Root-mean-square error is the root of the mean square error; C (p)=the Mallows statistic is for the degree of fitting of estimating regression model.SE is the average mistake.
The application of embodiment 4, wean swine rations metabolic energy level predictive equation
1, the acquisition of eight species diversity metabolic markers characteristics in the stripped blood plasma of weanling pig to be measured
1) 30 of the random acquisitions method according to embodiment 1 that the has neither part nor lot in embodiment 2 modelings stripped plasma sample of the weanling pig that different metabolic can level diet of feeding.
2) method according to step 1-3 among the embodiment 2 obtains 8 species diversity metabolic markers lysophosphatidyl cholines (18:3/0:0) in each sample, cool acyl group hemolytic phosphatid ylcholine (14:0/0:0), glycerophosphorylethanolamine (18:0/0:0), lysophosphatidyl choline (20:4/20:4), proline, phosphine acyl choline (18:2/0:0), tripeptides (arginyl phenylalanyl arginine) and glycerophosphorylethanolamine (18:1 (9Z)/0:0) and chromatographic peak area thereof; get lg value (namely carrying out the lg conversion); obtain the relative content data, the results are shown in Table 8.
The relative content data of table 8 wean to be measured pig blood plasma difference metabolic markers
2, the prediction of weanling pig daily ration metabolizable energy to be measured
6 predictive equations that the relative content data of the weanling pig blood plasma difference metabolic markers to be measured that table 8 is obtained are set up among the substitution embodiment 3 respectively calculate the predicted value of the metabolizable energy of daily ration that corresponding pig is fed.Calculate predicted value and the relative deviation between the actual value (RD%)=(predicted value-actual value)/actual value * 100% of the metabolizable energy of the daily ration of feeding simultaneously, the result is as shown in table 9.As shown in Table 9, only just can obtain relatively accurate metabolizable energy value by equation 1.Predicted value and actual value relative deviation are within ± 2%.And predict that by equation 2 to 6 can obtain result more accurately, the relative deviation of the predicted value of equation 6 is in ± 1%.In the table 9, ME is the metabolizable energy actual value of daily ration of feeding, and ME1-ME6 is respectively the metabolizable energy predicted value of the daily ration of feeding that obtains by the equation 1 among the embodiment 3 to equation 6.
The predicted value of 1 to 6 pair of weanling pig metabolizable energy to be measured of table 9 equation
3, brief summary
This test findings shows, according to the equation of setting up, can predict the metabolic energy level of daily ration that piglet is fed by blind sample pig blood plasma metabolic markers, and its predicated error is little, and the result has good application prospects accurately and reliably.
Claims (5)
1. quantitatively detect following 1)-6) in the product of arbitrary described compound group or method detect or the metabolizable energy value of auxiliary detection daily ration that pig is fed in application:
1) compound L LL;
2) compound L LL and compound MLC;
3) compound L LL, compound GP0 and compd A LL;
4) compound L LL, compound GP0, compd A LL and proline;
5) compound L LL, compound GP0, compd A LL, compound L PC, arginyl phenylalanyl arginine, compound GP1;
6) compound L LL, compound GP0, compd A LL, proline, compound L PC, arginyl phenylalanyl arginine, compound GP1;
The structural formula of described compound L LL is suc as formula shown in the I:
The structural formula of described compound L PC is suc as formula shown in the II:
The structural formula of described compound GP0 is suc as formula shown in the III:
The structural formula of described compd A LL is suc as formula shown in the IV:
The structural formula of described compound GP1 is suc as formula shown in the V:
The structural formula of described compound MLC is suc as formula shown in the VI:
2. one kind is detected or the method for the metabolizable energy value of auxiliary detection daily ration that pig is fed, and comprises following A)-F) at least one in the step:
A) with in the X1 substitution equation 1, obtain ME1; Described equation 1 is ME1=3252.2+79.8X1;
B) with in X1 and the X2 substitution equation 2, obtain ME2; Described equation 2 is ME2=3252.0+75.1X1-75.0X2;
C) with in X1, X3 and the X4 substitution equation 3, obtain ME3; Described equation 3 is ME3=3251.6+79.5 X1 – 53.7 X3 – 24.0 X4;
D) with in X1, X3, X4 and the X5 substitution equation 4, obtain ME4; Described equation 4 is ME4=3251.8+84.4 X1-45.8 X3 – 34.0 X4-42.5 X5;
E) with in X1, X3, X4, X6, X7 and the X8 substitution equation 5, obtain ME5; Described equation 5 is ME5=3251.6+95.5 X1 – 38.0 X3 – 20.8 X4 – 56.6 X6+52.7 X7-49.9 X8;
F) with in X1, X3, X4, X5, X6, X7 and the X8 substitution equation 5, obtain ME6; Described equation 6 is ME6=3251.7+96.0 X1-35.4 X3 – 28.4 X4 – 30.1 X5-46.6 X6+42.9 X7-40.2 X8;
Described ME1, ME2, ME3, ME4, ME5 and ME6 are the metabolizable energy value of daily ration that described pig to be measured is fed, and unit is kcal/kg;
Described X1 is the relative content of compound L LL described in the stripped blood plasma of pig to be measured;
Described X2 is the relative content of compound MLC described in the stripped blood plasma of pig to be measured;
Described X3 is the relative content of compound GPO described in the stripped blood plasma of pig to be measured;
Described X4 is the relative content of compd A LL described in the stripped blood plasma of pig to be measured;
Described X5 is the relative content of proline described in the stripped blood plasma of pig to be measured;
Described X6 is the relative content of compound L PC described in the stripped blood plasma of pig to be measured;
Described X7 is the arginic relative content of arginyl phenylalanyl described in the stripped blood plasma of pig to be measured;
Described X8 is the relative content of compound GP1 described in the stripped blood plasma of pig to be measured;
Described relative content is the lg value of getting the chromatographic peak area of respective compound in the stripped blood plasma of described pig to be measured.
3. method according to claim 2 is characterized in that:
Described chromatogram is liquid chromatography, and the testing conditions of described liquid chromatography is specific as follows: chromatographic column is the quick high separation chromatographic column of Agilent ZORBAX UHV (ultra-high voltage) C-18; Flowing, to be respectively A mutually be the aqueous solution of 0.1% formic acid mutually for volumn concentration, and B is the acetonitrile solution of 0.1% formic acid mutually for volumn concentration; Flow velocity is 0.3mL/min, and temperature is 40 ℃, and from the B phase gradient wash-out of 5%-95%, be 25min analysis time.
4. according to claim 2 or 3 described methods, it is characterized in that: before carrying out described liquid chromatography, the processing that the stripped blood plasma of described pig to be measured is comprised the steps: get described blood plasma and add among the extract A by the volume ratio of 1:4 and extract, obtain supernatant A, the volume ratio of pressing 1:1 in described supernatant A adds extract B, obtain supernatant B, described supernatant B is carried out described detection;
Described extract A is that methyl alcohol and acetonitrile are by the mixed solution of the volume ratio of 1:1;
Described extract B is that methyl alcohol and water are by the mixed solution of the volume ratio of 4:1.
In the claim 2-4 arbitrary described method in the preparation daily ration of pig or the application in the feed.
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