CN103245747B - Method for predicating metabolizable energy level of pig diets by metabonomics technology - Google Patents
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- Investigating Or Analysing Biological Materials (AREA)
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 applying 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, due to the application of the raw materials such as corn in manufacture alcohol etc., energy feed is caused to occur the phenomenon that shortage, price are more and more expensive.Therefore, how in feed, reasonably to utilize energy, both met the needs of piglet for energy, the waste of energy feed can be avoided again, just seem particularly important.
In Swine Production intensive at present, usually realize swinery according to the technological process of all-in and all-out and normally have enough to meet the need on a production line, the daily ration of identical metabolic energy level of feeding with a collection of pig.This production technology have ignored the difference of the individual and subpopulation of pig, and different pig farm and environment are on the impact 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 pedigrees and production performance; Equally, different nutrition supplies also can produce specific metabolite profile and production performance.That is same energy level is for different swinerys, and effective energy value may be different.Therefore, Different Individual or subpopulation, varying environment, different health status pig to daily ration metabolizable energy to need be different.If do not consider the variation of its effective energy value when preparing daily ration, daily ration actual can value and expecting can be worth between can be variant, if reality can value be greater than expectation can value, then can cause the waste of feed resource, even pollute environment; If actual can value be less than expectation can value, then can reduce or suppress the production performance of pig.
Animal nutrition research becomes more meticulous day by day, accurately determines that the requirement of nutrient has become domestic and international study hotspot to regulate and control growth of animal.In order to reduce feed cost, save feed resource and minimizing and to raise pigs the negative effect of industry to environment, in swine rations process for preparation, adopt the accurate effective energy value of raw material to be vital.Intracorporal method mensuration raw material effective energy value is wasted time and energy and cost is high, sets up ARIMAX model thus realizes by measuring material nutrient component and predict that the method that it can be worth also exists the shortcomings such as predictablity rate is low.
Metabolism group is the science of quantitative test biosome endogenous metabolism product species, quantity and Changing Pattern thereof, and it understands the situation of biosome and the result of variations of internal and external factor initiation by measuring end product of metabolism.Due to the pattern-recognition advantage on its high-throughout information science characteristic and basis thereof, by the correlation research of human nutriology's family expenses between the factor such as recipe, life style and metabolin spectral pattern, and attempt the new standard of the recognition mode of metabolism group as people's assessment of nutritional status, realize individual or subpopulation recipe recommendation.Due to the singularity of research object, cannot carry out artificial recipe operation, the qualification of mark depends on clinical nutrition study and analysis, and progress slowly.
Up to now, also do not utilize 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.), carry out the report of Accurate Prediction piglet diet effective energy value.
Summary of the invention
An object of the present invention is to provide and quantitatively detect following 1)-6) in the product of arbitrary described compound group or method detect or auxiliary detection pig the application of feeding in the metabolizable energy value of daily ration:
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 such as formula shown in I:
The structural formula of described compound L PC is such as formula shown in II:
The structural formula of described compound GP0 is such as formula shown in III:
The structural formula of described compd A LL is such as formula shown in IV:
The structural formula of described compound GP1 is such as formula shown in V:
The structural formula of described compound MLC is such as formula shown in VI:
The present invention also provide a kind of detect or auxiliary detection pig the method for metabolizable energy value of daily ration of feeding, comprise following A)-F) at least one in step:
A) X1 is substituted in equation 1, obtain ME1; Described equation 1 is ME1=3252.2+79.8 X1;
B) X1 and X2 is substituted in equation 2, obtain ME2; Described equation 2 is ME2=3252.0+75.1 X1-75.0X2;
C) X1, X3 and X4 are substituted in equation 3, obtain ME3; Described equation 3 is ME3=3251.6+79.5X1 – 53.7 X3 – 24.0 X4;
D) X1, X3, X4 and X5 are substituted in equation 4, obtain ME4; Described equation 4 is ME4=3251.8+84.4 X1-45.8 X3 – 34.0 X4-42.5 X5;
E) X1, X3, X4, X6, X7 and X8 are substituted in 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) X1, X3, X4, X5, X6, X7 and X8 are substituted in 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 X7-40.2X8;
Described ME1, ME2, ME3, ME4, ME5 and ME6 be described pig to be measured the metabolizable energy value of daily ration of feeding, unit is kcal/kg;
Described X1 is the relative content of compound L LL described in pig Blood plasma in vitro to be measured;
Described X2 is the relative content of compound MLC described in pig Blood plasma in vitro to be measured;
Described X3 is the relative content of compound GPO described in pig Blood plasma in vitro to be measured;
Described X4 is the relative content of compd A LL described in pig Blood plasma in vitro to be measured;
Described X5 is the relative content of proline described in pig Blood plasma in vitro to be measured;
Described X6 is the relative content of compound L PC described in pig Blood plasma in vitro to be measured;
Described X7 is the arginic relative content of arginyl phenylalanyl described in pig Blood plasma in vitro to be measured;
Described X8 is the relative content of compound GP1 described in pig Blood plasma in vitro to be measured;
Described relative content is the lg value of the chromatographic peak area getting respective compound in described pig Blood plasma in vitro to be measured.
In the above-mentioned methods, 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 C-18 of Agilent ZORBAX UHV (ultra-high voltage); Mobile phase be respectively A phase for volumn concentration be 0.1% first aqueous acid, B phase is the acetonitrile solution of the formic acid of 0.1% for volumn concentration; Flow velocity is 0.3mL/min, and temperature is 40 DEG C, and from the B phase gradient wash-out of 5%-95%, analysis time is 25min.
In the above-mentioned methods, before carrying out described chromatogram, the Blood plasma in vitro of described pig to be measured is carried out the process comprised the steps: get described blood plasma and add in extract A by the volume ratio of 1:4 and extract, obtain supernatant A, extract B is added by the volume ratio of 1:1 in described supernatant A, 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 above-mentioned methods, the kind of pig described in embodiments of the invention be Duroc × length white × great Bai ternary grower pigs, the body weight of described pig before described daily ration of feeding is 13.4 ± 2.1kg, predict described pig the metabolizable energy value of daily ration of feeding time, the feeding cycle of described daily ration is 28 days.
The present invention protects the application of above-mentioned arbitrary described method in the daily ration or feed of preparation pig.
Described metabolizable energy can deduct the fecal energy of pig with feed, urinate the energy of energy and alimentary canal combustible gas.Feed can refer to the energy that feed produces when burning completely and produce end-product water and carbon dioxide.
The present invention is by the analysis of blood plasma end product of metabolism, have found the blood plasma metabolic markers and Changing Pattern thereof that associate with daily ration metabolizable energy, and according to the metabolite markers prediction piglet fed in different metabolic energy level diet weanling pig blood plasma effective metabolic energy level of daily ration of feeding, set up the dynamic regression predictive equation of blood plasma metabolic markers and effective energy value, wherein, the daily ration metabolizable energy predicted value using equation with one unknown quantity provided by the present invention to obtain and the relative deviation of actual value are within ± 2%, the daily ration metabolizable energy predicted value using seven yuan of equations provided by the present invention to obtain and the relative deviation of actual value are within ± 1%.The present invention, in time evaluating nutrition condition and optimization of C/C composites, meets the nutritional need of pig under varying environment, physiology and pathological conditions, improves pig production efficiency and have important theory and practice meaning.
Accompanying drawing explanation
Fig. 1 is the blood plasma metabolism group principal component analysis (PCA) shot chart of weanling pig of different metabolic energy level diet of feeding.Wherein, icon 1-5 represents the processed group result that daily ration metabolic energy level of feeding is 3150,3200,3250,3300 and 3350kcal/kg respectively.
Fig. 2 is blood plasma metabolism group partial least square method discriminatory analysis (PLS-DA) shot chart of weanling pig of different metabolic energy level diet of feeding.Wherein, icon 1-5 represents the processed group result that daily ration metabolic energy level of feeding is 3150,3200,3250,3300 and 3350kcal/kg respectively.
Embodiment
The experimental technique used in following embodiment if no special instructions, is conventional method.
Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
Embodiment 1, different daily ration metabolic energy level are on the impact of piglet growth performance
1, experimental animal
Test select healthy Duroc × length white × great Bai ternary grower pigs totally 180, male and female half and half, average Weaning Age is 28 days, and transition formally starts after 1 week to test.Test original body mass is 13.4 ± 2.1kg, and with original body mass, be divided into 5 process, each process 6 repetition according to randomized block experiment design, often circle is a repetition, each repetition 6 pigs.Male and female is separately raised.
2, feeding and management
Adopt all-in and all-out feeding and management pattern, pig house temperature controls at 24-27 DEG C, and illumination program is that 12h illumination/12h is dark.Each circle (1.5 × 3.0m
2) be all furnished with 1 nipple-shaped drinker and 2 feed bunks.Net bed is raised, periodic cleaning ight soil.Powder is fed, free choice feeding and drinking-water.Immune expelling parasite is carried out according to pig farm Routine Management program.
3, daily ration is tested
Experimental basis daily ration is corn-soybean meal, uses the corn of different proportion and soybean oil to adjust daily ration metabolisable energy content.5 process daily ration metabolic energy level are respectively 3150,3200,3250,3300 and 3350kcal/kg, and standard ileal apparent digestible lysine and metabolizable energy ratio (SID Lys:ME) keep 3.78g/Mcal.Adjust other several essential amino acid (methionine, threonine and tryptophane) level according to ideal protein model (swine rearing standard, 2004) simultaneously, itself and lysine ratio are consistent.Add 0.25% chrome green as indicator.Diet Formula and nutrient calculation value are in table 1.
4, data acquisition
Experimental period is 28 days.On-test with at the end of, adopt electronic platform scale to weigh piglet whose body weight and feed relative respectively, calculate average daily gain, average daily ingestion amount and weightening finish material consumpting ratio (or feed efficiency).After off-test, piglet taboo raises 12h, 3 piglets are selected at random from often enclose, gather vena cava anterior blood, Blood Sample Collection, in liquaemin anti-freezing vacuum test tube, is placed in ice chest and is transported to laboratory, 4 DEG C, the centrifugal 15min of 3000rpm, separated plasma, obtain plasma samples ex vivo, and packing is stored in-80 DEG C of refrigerators.
5, data statistics
The test figure participating in blood sampling piglet adopts the general linear model (general linear models, GLM) in SAS V8.02 statistical software to carry out statistical study according to randomized block experiment design.Take original body mass as test site group, total model comprises daily ration effect, block effect and stochastic error, as follows:
Y
ij=μ+T
ij+R
ij+ξ
ij
(i=1,2…,a,j=1,2…,b)
Wherein Y
ijfor observed value, μ is population mean, T
ijfor daily ration effect, R
ijfor block effect, ξ
ijfor stochastic error.A is process number (being 5 in this test), and b is block number (being 6 in this test).If significant difference between each process, then test with Tukey Multiple range test.The result average of least square method and average represent by mistake, and P<0.05 is significant difference.Linear model and conic model is adopted to analyze daily ration different metabolic energy level to the impact of piglet growth performance.
6, result
Duration of test swinery general health is good.Daily ration different metabolic energy level on the impact of piglet growth performance in table 2.Compared with being the processed group of 3350kcal/kg with daily ration metabolic energy level of feeding, daily ration metabolic energy level of feeding be 3150 and the piglet final body of processed group of 3300kcal/kg heavily significantly improve (P<0.05), daily ration metabolic energy level of feeding be 3150,3200 and the piglet average daily gain of processed group of 3300kcal/kg significantly improve.Along with the rising of daily ration metabolic energy level, the average daily ingestion amount of piglet presents the trend of reduction, daily ration metabolic energy level of feeding is that the processed group of 3150kcal/kg is the highest, and daily ration metabolic energy level of feeding is minimum (linearly, the P<0.05 of the processed group of 3350kcal/kg; Secondary, P<0.05).Daily ration metabolic energy level does not make significant difference (P>0.05) to weight gain of piglets material consumpting ratio and feed efficiency.
Table 2 daily ration different metabolic energy level is for the impact of piglet growth performance
Note:
1average by mistake.
2process refers to the result of Multiple range test, the effect of linear and secondary in a few days grain different metabolic energy level.With in a line, female different expression significant difference (P<0.05) of shoulder marking-up.
7, brief summary
Weanling pig, due to its daily ration be transitioned into solid-state feed from liquid pig breast, turn group feeding support and factor such as psychology change etc. cause stress, add that the feed intake of weanling pig is on the low side, sometimes even there will be the negative growth of body weight, mainly body fat loses larger.Many affect weaned piglet after growth performance trophism factor in, Dietary ME is particularly important.Therefore suitably to improve the material composition of high digestibility and high-energy ratio in weanling pig daily ration, reduce indigestibility and the low feedstuff ratio of energy.The average daily ingestion amount of the metabolic energy level appreciable impact piglet of daily ration, piglet can carry out regulate intake according to the concentration of energy in daily ration, keeps the constant of Energy intaking amount.In this test, metabolizable energy and apparent digestible lysine (the first limiting amino acids of pig) keep constant ratio, even if high-energy group piglet feed intake declines, do not have influence on the actual intake of piglet amino acid and metabolizable energy, simultaneously, piglet daily gain is also on the low side compared with other groups, thus does not make significant difference to feed efficiency.
Under this test condition, the average daily ingestion amount of comprehensive piglet is comparatively large, the suitable daily ration metabolizable energy requirement of average daily gain and the smaller daily ration metabolizable energy of weightening finish feed consumption and piglet is 3300kcal/kg, now economic benefit is maximum, namely expends minimum feed and obtains maximum weightening finish.
The test daily ration of table 1 piglet different metabolic energy and trophic level
1premix is pressed 1% of daily ration and is added, for every kilogram of daily ration provides: vitamin A, 12,000IU in daily ration; 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.
Embodiment 2, screening based on the dietary digestibility of energy label of blood plasma metabolism group profile
1, the process of sample
In thawing on ice after the piglet plasma samples ex vivo of embodiment 1 is taken out from-80 DEG C 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, extracts after 1 hour at-20 DEG C, at 4 DEG C, centrifugal 10min under 13000rpm.Careful absorption supernatant 200 μ L is placed in new centrifuge tube, dries up under normal temperature with nitrogen, and redissolves in the methanol solution of 200 μ L 80%.After concussion 5s, centrifuging and taking supernatant again, is placed in sample injection bottle, 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 μm), mobile phase be respectively A phase for volumn concentration be 0.1% first aqueous acid, B phase is the acetonitrile solution of the formic acid of 0.1% for volumn concentration; Flow velocity 0.3mL/min, temperature 40 DEG C, from the B phase gradient wash-out of 5%-95%, analysis time is 25min.
Mass spectrographic condition: ion gun is electron spray holotype ESI+, dry gas temperature 350 DEG C, dry gas flow velocity 12L/min, capillary voltage 3500V, cracked voltage 150V, acquisition quality scope 60-1000m/z, acquisition rate 2spectra/s.
3, data processing
Detect through the HPLC Q-TOF MS of step 2, obtain the metabolism group profile raw data of each sample, use MassHunter Qualitative Analysis software (version B.03.01, Agilent company), utilize the metabolism group profile raw data of characterization of molecules extraction algorithm to all samples to carry out the calibration of background deduction, spectrum peak accurate mass number and retention time, extract the characteristic of the retention time of each sample, mass-to-charge ratio and ion-intensity values.Data importing Mass Profiler Professional(is called for short MPP, version number B.02.00, Agilent company), through the filtration of peak match, alignment, default parameter with after correcting, the compound filtered out is normalized (i.e. lg conversion) again, obtains the data set correcting rear metabolin characterization of molecules.Carry out packet and screening, statistical study, the analysis of change multiple again, after obtaining the biomarker of difference, then the inspection of extracting chromatography of ions figure (EIC) is carried out to data, get rid of the result of false sun (the moon) property.
4, principal component analysis (PCA) (PCA)
The data utilizing MPP software to obtain step 3 find the linear relationship between original variable by principal component analysis (PCA), form new variable.These major components are by arranging the reserving degree size order of raw data variance, orthogonal each other.PCA shot chart as shown in Figure 1.Fig. 1 result shows, and the piglet plasma sample of the processed group of different metabolic of feeding energy level diet can obviously cluster be to together, and first principal component and Second principal component, can explain the group difference of 44.64% and 24.27% respectively.This illustrates the piglet of different metabolic energy level diet of feeding, and the metabolite profile of its blood plasma exists significant difference.
5, the foundation of Partial Least-Squares Regression Model and prediction
Use the partial least square method discriminatory analysis (PLS-DA) in MPP software, set up multi-parameters model and sample is predicted.The metabolism modal data processed through step 3 randomly drawing 5 samples from each processed group carries out modeling as the sample investigated for Modling model (training set), select variable sample classification to appreciable impact, the metabolism modal data processed through step 3 of remaining sample to be verified model as the checking sample (test set) of multiple cross checking screening model and predicts, finally obtaining reliable and stable pattern recognition model.From five groups of blood plasma PLS-DA shot charts (Fig. 2), the blood plasma metabolism spectrum of the grower pigs of five kinds of different metabolic energy level diet of feeding can distinguish completely, is gathered 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, does not have the grouping of wrong identification to occur.
Table 3 utilizes recognition capability and the predictive ability of model after PLS-DA modeling
6, the qualification of blood plasma difference metabolic markers
From step 5, on grouping, there is the existing larger covariance coefficient of final selection in the variable of appreciable impact, have again the variable of larger related coefficient to be characterize the mark being somebody's turn to do " metabolic energy level model ".The compound exact mass number obtained by HPLC Q-TOF MS mass spectral results carries out the generation of molecular formula, simultaneously at METLIN, HMDB, KEGG, the compound structure that the database retrievals such as LIPIDMAPS are possible, to qualified compound, the MS/MS carried out under different cracking energy analyzes, the second order ms figure of acquisition and candidate markers are carried out binding analysis, compare with the existing standard items second order ms of part simultaneously, finally identify following 10 kinds of compounds (table 4) relevant to daily ration metabolic energy level: amino acid and polypeptide class: lysine (hereinafter referred to as LYS), phenylalanine (hereinafter referred to as PHE), proline (hereinafter referred to as PRO) and arginyl phenylalanyl arginine (hereinafter referred to as APA), lipid: lysophosphatidyl choline (18:3/0:0) (hereinafter referred to as ALL), glycerophosphorylethanolamine (18:0/0:0) (hereinafter referred to as GP0), phosphine phosphatidylcholine (18:2/0:0) (hereinafter referred to as LPC), lysophosphatidyl choline (20:4/20:4) (hereinafter referred to as ALL), glycerophosphorylethanolamine (18:1 (9Z)/0:0) (hereinafter referred to as GP1) and cool acyl group Lysophosphatidylcholone (14:0/0:0) (hereinafter referred to as MLC).
Table 4 feed different metabolic energy level diet weanling pig blood plasma in the qualification result of endogenous difference metabolic markers
Note: W
l, W
2and W
3be respectively metabolic markers in major component 1,2, the weighted value in 3.
In table 4, the structural formula of described compound L LL is such as formula shown in I:
The structural formula of described compound L PC is such as formula shown in II:
The structural formula of described compound GP0 is such as formula shown in III:
The structural formula of described compd A LL is such as formula shown in IV:
The structural formula of described compound GP1 is such as formula shown in V:
The structural formula of described MLC is such as formula shown in VI:
7, brief summary
The weanling pig of different metabolic of searching for food energy level diet, there is significant difference in the metabolite profile in blood plasma.Principal component analysis (PCA) shows with partial least square method discriminatory analysis result: the piglet blood plasma of five kinds of daily rations of searching for food can obviously cluster to together with, blood plasma metabolism spectrum can distinguish completely.Discrimination model recognition capability and the predictive ability of foundation are high, can with accurately differentiating different daily ration metabolic energy level.Meanwhile, identify the closely-related 10 species diversity metabolic markers with 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 object of the present embodiment is the analysis by blood plasma end product of metabolism, find out the metabolic markers and Changing Pattern thereof that associate with nutrition parameters with production performance, inquire into and how to predict the effective metabolic energy level of piglet according to the metabolite markers of searching for food in different metabolic energy level diet weanling pig blood plasma, set up the dynamic regression predictive equation of blood plasma metabolic markers and effective energy value, attempt by means of only one or more blood plasma metabolic markers, carry out the effective energy value of Accurate Prediction piglet, for evaluating nutrition condition and optimization of C/C composites in time, meet varying environment, the nutritional need of pig under physiology and pathological conditions, improve pig production efficiency and there is important theory and practice meaning.
1, the relative content of difference metabolic markers
The chromatographic peak area of 10 kinds of blood plasma difference metabolic markers of sample in each processed group obtained according to step 3 in embodiment 2 is got lg value (namely carrying out lg conversion), namely obtains 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 software, during significant difference, carry out Tukey Multiple range test.Various blood plasma metabolic markers at 5 kinds of daily ration metabolic energy level condition allowance below nominal size heteropoles significantly (P<0.01).
2, the correlation analysis of difference metabolic markers and daily ration metabolizable energy value
The relative content data acquisition JMP statistical software Multivariate method of each difference metabolic markers is carried out correlation analysis, and result is as shown in table 6.Result shows, except glycerophosphorylethanolamine (18:0/0:0), proline and cool acyl group Lysophosphatidylcholone (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 the positive correlation of daily ration metabolic energy level, especially lysophosphatidyl choline (18:3/0:0) and daily ration metabolic energy level correlativity very high (0.828); Blood plasma phosphine phosphatidylcholine (18:2/0:0) and arginyl phenylalanyl arginine-level and daily ration metabolic energy level also have the correlativity of nearly 60%.
Table 5 is fed the relative content of weanling pig blood plasma difference metabolic markers of different metabolic energy level diet
Note: P<0.05 carries out Tukey Multiple range test, with in a line, and female different expression significant difference (P<0.05) of shoulder marking-up.
The relative content of table 6 difference metabolic markers and the related coefficient of daily ration metabolic energy level
Note: ME is the metabolizable energy value of daily ration.
3. the regretional analysis of blood plasma metabolic markers and daily ration metabolizable energy
The stepwise in JMP 9 is adopted to carry out simple regression analysis and stepwise regression analysis showed (stepwiseregression analysis), to study the relation of daily ration metabolic energy level and difference metabolic markers.After table 7 lists and carries out regretional analysis to daily ration metabolic energy level and blood plasma difference metabolic markers, the prediction regression equation of the metabolizable energy of foundation.P value be greater than 0.05 equation unlisted.Lysophosphatidyl choline (18:3/0:0) can set up the regression equation with one unknown of the effective metabolic energy level of piglet diet as unique predictor, equation is: ME1=3252.2+79.8X1, its coefficient of determination (Adj R revised
2) be 0.682, predictive ability is better.In addition, the foundation of multiple regression equation improves the accuracy of predictive equation, sets up metabolizable energy predictive equation, make Adj R by 2 species diversity metabolic markers to 7 kinds of metabolic markers
20.863 is increased to from 0.784.
Binary is respectively to seven yuan of equations: 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 X7-49.9X8(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 described in pig Blood plasma in vitro (i.e. metabolic markers) LLL, MLC, GP0, ALL, PRO, LPC, APA and GP1.
4. brief summary
This test findings shows, blood plasma difference metabolic markers can must predict weanling pig daily ration metabolic energy level very well, and unitary predictive equation is ME=3252.2+79.8 X1(Adj R
2=0.682), 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, (AdjR
2=0.863).
The linear regression prediction equation of table 7 weanling pig daily ration metabolic energy level
Note: Adj R
2for the coefficient of determination revised, be used for evaluating the goodness of fit of regression equation.Root-mean-square error is the root of the mean squareerror; C (p)=the Mallows statistic, for evaluating the degree of fitting of regression model.SE is that average misses.
The application of embodiment 4, wean swine rations metabolic energy level predictive equation
The acquisition of eight species diversity metabolic markers characteristics in the Blood plasma in vitro of 1, weanling pig to be measured
1) random acquisition 30 has neither part nor lot in the plasma samples ex vivo of weanling pig of the different metabolic energy level diet of feeding according to the method for embodiment 1 of embodiment 2 modeling.
2) 8 species diversity metabolic markers lysophosphatidyl cholines (18:3/0:0), cool acyl group Lysophosphatidylcholone (14:0/0:0), glycerophosphorylethanolamine (18:0/0:0), lysophosphatidyl choline (20:4/20:4), proline, phosphine phosphatidylcholine (18:2/0:0), tripeptides (arginyl phenylalanyl arginine) and glycerophosphorylethanolamine (18:1 (9Z)/0:0) and chromatographic peak area thereof in each sample is obtained according to the method for step 1-3 in embodiment 2; get lg value (namely carrying out lg conversion); obtain relative content data, the results are shown in Table 8.
The relative content data of table 8 wean Swine plasma to be measured difference metabolic markers
2, the prediction of weanling pig daily ration metabolizable energy to be measured
The relative content data of the weanling pig blood plasma difference metabolic markers to be measured obtained by table 8 substitute into 6 predictive equations set up in embodiment 3 respectively, calculate corresponding pig the predicted value of metabolizable energy of daily ration of feeding.Calculate relative deviation (RD%)=(predicted value-actual value)/actual value × 100% between the predicted value of the metabolizable energy of daily ration of feeding and actual value, result is as shown in table 9 simultaneously.As shown in Table 9, just relatively accurate metabolizable energy value can be obtained by means of only equation 1.Predicted value and actual value relative deviation are within ± 2%.And predicted by equation 2 to 6, can obtain result more accurately, the relative deviation of the predicted value of equation 6 is within ± 1%.In 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 obtained by equation 1 to the equation 6 in embodiment 3.
Table 9 equation 1 to 6 is to the predicted value of weanling pig metabolizable energy to be measured
3, brief summary
This test findings shows, according to the equation set up, by blind sample Swine plasma metabolic markers can predict piglet the metabolic energy level of daily ration of feeding, its predicated error is little, and result accurately and reliably, has good application prospect.
Claims (4)
1. detect or auxiliary detection pig the method for metabolizable energy value of daily ration of feeding, comprise following A)-F) step:
A) X1 is substituted in equation 1, obtain ME1; Described equation 1 is ME1=3252.2+79.8X1;
B) X1 and X2 is substituted in equation 2, obtain ME2; Described equation 2 is ME2=3252.0+75.1X1 – 75.0X2;
C) X1, X3 and X4 are substituted in equation 3, obtain ME3; Described equation 3 is ME3=3251.6+79.5X1 – 53.7X3 – 24.0X4;
D) X1, X3, X4 and X5 are substituted in equation 4, obtain ME4; Described equation 4 is ME4=3251.8+84.4X1 – 45.8X3 – 34.0X4 – 42.5X5;
E) X1, X3, X4, X6, X7 and X8 are substituted in equation 5, obtain ME5; Described equation 5 is ME5=3251.6+95.5X1 – 38.0X3 – 20.8X4 – 56.6X6+52.7X7 – 49.9X8;
F) X1, X3, X4, X5, X6, X7 and X8 are substituted in equation 5, obtain ME6; Described equation 6 is ME6=3251.7+96.0X1 – 35.4X3 – 28.4X4 – 30.1X5 – 46.6X6+42.9X7 – 40.2X8;
Described ME1, ME2, ME3, ME4, ME5 and ME6 be pig to be measured the metabolizable energy value of daily ration of feeding, unit is KCAL/KG;
Described X1 is the relative content of compound L LL in pig Blood plasma in vitro to be measured;
Described X2 is the relative content of compound MLC in pig Blood plasma in vitro to be measured;
Described X3 is the relative content of compound GP0 in pig Blood plasma in vitro to be measured;
Described X4 is the relative content of compd A LL in pig Blood plasma in vitro to be measured;
Described X5 is the relative content of proline in pig Blood plasma in vitro to be measured;
Described X6 is the relative content of compound L PC in pig Blood plasma in vitro to be measured;
Described X7 is the arginic relative content of arginyl phenylalanyl in pig Blood plasma in vitro to be measured;
Described X8 is the relative content of compound GP1 in pig Blood plasma in vitro to be measured;
The structural formula of described compound L LL is such as formula shown in I:
Formula I;
The structural formula of described compound L PC is such as formula shown in II:
Formula II;
The structural formula of described compound GP0 is such as formula shown in III:
Formula III;
The structural formula of described compd A LL is such as formula shown in IV:
Formula IV;
The structural formula of described compound GP1 is such as formula shown in V:
Formula V;
The structural formula of described compound MLC is such as formula shown in VI:
Formula VI;
Described relative content is the LG value of the chromatographic peak area of respective compound in the Blood plasma in vitro getting described pig to be measured.
2. method according to claim 1, 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 C-18 of Agilent ZORBAX UHV (ultra-high voltage); Mobile phase be respectively A phase for volumn concentration be 0.1% first aqueous acid, B phase is the acetonitrile solution of the formic acid of 0.1% for volumn concentration; Flow velocity is 0.3ML/MIN, and temperature is 40 DEG C, and from the B phase gradient wash-out of 5%-95%, analysis time is 25MIN.
3. method according to claim 2, it is characterized in that: before carrying out described liquid chromatography, the Blood plasma in vitro of described pig to be measured is carried out the process comprised the steps: get described blood plasma and add in extract A by the volume ratio of 1:4 and extract, obtain supernatant A, extract B is added by the volume ratio of 1:1 in described supernatant A, 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.
4. the application of arbitrary described method in the daily ration or feed of preparation pig in claim 1-3.
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