CN101285828B - Rapid quantitative analysis method for animals skeletal muscle fiber - Google Patents
Rapid quantitative analysis method for animals skeletal muscle fiber Download PDFInfo
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Abstract
The invention discloses a method for rapidly and quantitatively analyzing skeletal muscle fibers of an animal. The method comprises the following steps of: 1) selecting skeletal muscle separated from an animal body to fabricate a frozen section; 2) carrying out succinate dehydrogenase dyeing for the frozen section in the step 1); 3) taking a picture for the dyed section in the step 2), saving the picture, and then quantitatively analyzing the picture by computer image processing and analyzing software. The succinate dehydrogenase dyeing is an improvement based on the prior succinate dehydrogenase dyeing technology. The computer image processing and analyzing software is software of Image Pro-plus 5.02 version. The method for rapidly and quantitatively analyzing skeletal muscle fibers of an animal is capable of screening out individuals with good meat quality from the viewpoint of muscle fiber classification.
Description
Technical field
The present invention relates to the method for fast quantitative analysis animals skeletal muscle fiber.
Background technology
The animal muscle quality is actually a complicacy and changeable notion, and meat relates to sense index (yellowish pink, marble grain, mouthfeel etc.), physical index (shearing force, percentage of water loss, cold cuts rate), biochemical indicator many aspects such as (fat content, imp content, phosphatide, muscle fiber types, muscle fibre density, muscle fibre area, diameter of muscle fiber etc. between pH value, flesh).Increasing research shows that tender degree is the determinative and the most important organoleptic feature of leading meat, is the good and bad important indicator of evaluation meat quality.
Animals skeletal muscle muscle quality also is one of key factor of the tender degree of influence, no matter butcher live-weight size or age in days sooner or later, all is that the muscle fibre thin person's meat of healing is tenderer, and the muscle fibre number in the muscle bundle is many more, and muscle fibre is thin more, and correspondingly meat matter is also just more delicate.Relevant research reports that also diameter of muscle fiber is thin more, and myofibrillar quantity is many more in the unit muscle area of section, and it is also just more little to cut off the required shearing force of muscle.The muscle fibre ratio also influences the tender degree of meat, and muscle fibre can be divided into fiber of red muscle, white muscle fiber and intermediate fiber according to the difference of its metabolic way.The fiber of red muscle diameter is thinner, and the fiber number of unit area is many, and myoglobin content is abundant, and the ability of metabolism and depot lipid is stronger, contains more lipid material, mainly with the energy supply of aerobic oxidation form, has higher atpase activity; The white muscle fiber diameter is bigger, the negligible amounts of unit area, and it is more to contain glycogen, mainly with the energy supply of glycolysis form.Therefore, two kinds of muscle fibres can make a distinction according to atpase activity, NADH enzymatic activity (Y.ONO, 1993) or the activity of succinate dehydrogenase.The livestock and poultry of growth maybe be because the too fast speed of growth causes the morphologic change of muscle fast; Thicker fibre diameter and glycolysis fiber (white muscle fiber) number increase; Cause lower proteolysis potential; Butcher these characteristics of back and can cause that muscle becomes stiff quickly, form color pale, be the meat products of low, the tender degree difference of waterpower, be unfavorable for deep processing.
In order in the performance measurement process, to record the muscle fibre index fast, must carry out somatotype, dyeing and fiber measurement to muscle fibre to reach the purpose of meat evaluation.To myofibrillar somatotype, generally all be to utilize succinate dehydrogenase (SDH) dyeing, succinate dehydrogenase is the important dehydrogenasa in the tricarboxylic acid cycle, can be with glycometabolic intermediate product oxidative dehydrogenation.NBT (NBT) is a leuco dye, can be reduced to blue first
can impel have engulf, the neutrophil cell glycometabolism ability of sterilizing ability strengthens.The hydrogen taken off of succinate dehydrogenase in glycometabolism can reduce NBT, presents blue reaction particles diformazan
and is deposited in the kytoplasm.Therefore through SDH dyeing, can be according to the height of the enzymatic activity of the succinate dehydrogenase of different fibers, the difference of reduction NBT degree, the blue particles that form different amounts are deposited in the muscle fibre.Like this, can distinguish different muscle fiber typeses according to the depth degree of blueness reaction.Fiber of red muscle contains higher SDA, shows bluish violet; White muscle fiber succinate dehydrogenase enzymatic activity is very low, is shown as not painted or very light painted; And the general muscle fibre of succinate dehydrogenase enzymatic activity, coloring degree is between the two color, is intermediate fiber.
In present succinate dehydrogenase (SDH) the staining technique flow process, before tissue and the dyeing liquor reaction that contains NBT (NBT), need with neutral formalin effect more than 2 hours, the form of tissue is fixed.A series of processes such as dyeing will be experienced the gradient ethanol dehydration afterwards, and xylene is transparent.
Too strong in the past the most subjectivity of method aspect the muscle fibre analysis; It is mainly reflected in artificial counting behind the microscopic photography; Perhaps artificially measure the major axis and the minor axis of fiber cross section (being similar to ellipse) and roughly estimating its area; This method not only wastes time and energy, and subjectivity is strong, and accuracy is low.
Summary of the invention
The method that the purpose of this invention is to provide the fast quantitative analysis animals skeletal muscle fiber.
The method of fast quantitative analysis animals skeletal muscle fiber provided by the present invention comprises the steps:
1) gets the skeletal muscle of the animal of exsomatizing, process frozen section;
2) frozen section in the step 1) is carried out succinate dehydrogenase dyeing;
3) to step 2) section after the dyeing takes pictures, and preserves picture, with computing machine image processing and analyzing software picture carried out quantitative test then.
Wherein, the dyeing of the succinate dehydrogenase in the said method comprises the steps:
A) with said frozen section in the succinate dehydrogenase dyeing liquor, hatch 40min for 37 ℃;
B) with the fixing 10min in 10% neutral formalin of the section after the dyeing in the step a);
C) ethanol of the section after fixing in the step b) with gradient concentration (75%, 85%, 95% and 100%) is dewatered step by step;
D) using volume ratio is the section after the ethanol dehydration in 1: 2 to 2: 1 ethanol and the transparent step c) of xylene mixed liquor;
E) with the transparent section of handling through step d) of 100% xylene.
In the said method, adopt Image Pro-plus 5.02 version softwares that said picture is carried out quantitative test.
Above-mentioned animal specifically can be chicken.
Method of the present invention can be used in the meat of poultry or fowl is identified.
Said fowl specifically can be chicken.
The method of fast quantitative analysis animals skeletal muscle fiber of the present invention; At first take liquid nitrogen frozen to handle the skeletal muscle sample; Liquid nitrogen frozen is handled and can be kept the inner complete original form of muscle on the one hand, measures thereby make it can not be out of shape influence because of time, factor such as stressed, avoids the water loss that makes muscle inner on the other hand; Cause muscular atrophy; Avoid the inner moisture nucleation of muscle to form ice crystal simultaneously, ice crystal destroys bigger to the muscle fibre form, directly influence the measurement of diameter of muscle fiber and density; Adopt frozen section technique preparation section then, frozen section technique can keep organizing original form, and myofibrillar testing index is almost identical with its index in animal body in the section; Frozen section speed is fast in addition; Stressed little when being organized in section, be beneficial to the quantitative test in later stage, and adopt the serial section of unified standard; Make to have very high similarity between the section, be beneficial to a large amount of sample statistics and correlation analysis to each other; Adopt improved succinate dehydrogenase colouring method that section is dyeed once more; In present succinate dehydrogenase (SDH) the staining technique flow process, before dyeing, section needs long formaldehyde fixed; Dyeing time is longer; And improved succinate dehydrogenase colouring method will reach fixing this step before 2 hours the dyeing originally and change fixing after the dyeing into, and its time was controlled in 10 minutes, and the time with dyeing liquor painted this step of reaction of containing NBT (NBT) also shortened to present 40 minutes by original more than 1 hour in addition; And in this step of gradient ethanol dehydration after dyeing; Remove 50% and 60% these two gradients for the whole structure did not influence, dyeing time shortens greatly under the constant situation of bulk dyeing effect like this, and to the Color did not influence; At last; After dyeing, adopt ImagePro-Plus software quantitative test picture, this method has replaced loaded down with trivial details measurement manually, makes measuring speed improve greatly; Also avoided simultaneously the error that manually-operated brought; Utilization has loaded the software of new macros, can in single job, accomplish the analysis of a plurality of indexs such as myofibrillar classification, counting, diameter, area and density of a sectioning image, obtains data after the analysis; The analysis that can combine with conventional meat indexs such as shearing force, percentages of water loss makes meat assessment of data complete and accurate more.
Method through fast quantitative analysis animals skeletal muscle fiber of the present invention; On the one hand; Can obtain detailed muscle fibre characteristic somatotype and survey record fast, these data can be described the muscle fibre characteristic more in detail, exactly, the good and bad level of reflection meat; Thereby objectively the meat quality of working sample is made an appraisal, utilize with subculture seed selection and the hybridization of instructing kind.On the other hand, avoid stochastic error.The method of fast quantitative analysis animals skeletal muscle fiber of the present invention has solved quick film-making and has analyzed two big key issues automatically, makes that the acquisition of muscle fibre data is more easy.Fiber of red muscle is abundant just to mean that muscle fibre is more very thin, and it also more helps keeping the moisture in the muscle fibre, makes the more fine and smooth succulence of muscle.Therefore, the method for fast quantitative analysis animals skeletal muscle fiber of the present invention can be from the good individuality of angle screening meat of muscle fibre classification.
Description of drawings
Fig. 1 is myofibrillar somatotype standard
RF: fiber of red muscle, MF: osculant, WF: white muscle fiber
Fig. 2 is the division of muscle fiber types
A: white muscle fiber, b: fiber of red muscle
Fig. 3 is western Shandong cockfighting and Qingyuan City dissimilar musculus pectoralis superficialis of fiber crops chicken types of organization number percent
Fig. 4 is the western Shandong cockfighting of RMW type and the musculus pectoralis superficialis muscle fiber types ratio of Qingyuan City fiber crops chicken
Fig. 5 is the western Shandong cockfighting of RMW type and the musculus pectoralis superficialis diameter of muscle fiber of Qingyuan City fiber crops chicken
Fig. 6 is dissimilar western Shandong cockfighting and the body weight of Qingyuan City fiber crops chicken
1: the body weight of all western Shandong cockfighting; 2: the body weight of all Qingyuan City fiber crops chickens; 3: the body weight of the hen in all western Shandong cockfighting and Qingyuan City fiber crops chicken; 4: the body weight of the cock in all western Shandong cockfighting and Qingyuan City fiber crops chicken; 5: the body weight of RMW type chicken in all western Shandong cockfighting and Qingyuan City fiber crops chicken; 6: the body weight of MW type chicken in all western Shandong cockfighting and Qingyuan City fiber crops chicken; 7: the body weight of W type chicken in all western Shandong cockfighting and Qingyuan City fiber crops chicken.
Fig. 7 is the shearing force of the musculus pectoralis superficialis tissue of dissimilar western Shandong cockfighting and Qingyuan City fiber crops chicken
1: the shearing force of the musculus pectoralis superficialis tissue of all western Shandong cockfighting; 2: the shearing force of the musculus pectoralis superficialis tissue of all Qingyuan City fiber crops chickens; 3: the shearing force of the musculus pectoralis superficialis tissue of RMW type chicken in all western Shandong cockfighting and Qingyuan City fiber crops chicken; 4: the shearing force of the musculus pectoralis superficialis tissue of MW type chicken in all western Shandong cockfighting and Qingyuan City fiber crops chicken; 5: the shearing force of the musculus pectoralis superficialis tissue of W type chicken in all western Shandong cockfighting and Qingyuan City fiber crops chicken.
Fig. 8 is the percentage of water loss of the musculus pectoralis superficialis tissue of dissimilar western Shandong cockfighting and Qingyuan City fiber crops chicken
1: the percentage of water loss of the musculus pectoralis superficialis tissue of all western Shandong cockfighting; 2: the percentage of water loss of the musculus pectoralis superficialis tissue of all Qingyuan City fiber crops chickens; 3: the percentage of water loss of the musculus pectoralis superficialis tissue of RMW type chicken in all western Shandong cockfighting and Qingyuan City fiber crops chicken; 4: the percentage of water loss of the musculus pectoralis superficialis tissue of MW type chicken in all western Shandong cockfighting and Qingyuan City fiber crops chicken; 5: the percentage of water loss of the musculus pectoralis superficialis tissue of W type chicken in all western Shandong cockfighting and Qingyuan City fiber crops chicken.
Embodiment
One, the skeletal muscle fiber of the cockfighting of quantitative test western Shandong and Qingyuan City fiber crops chicken
The western Shandong cockfighting is a kind of native hen kind of China, belong to enjoying type, but build is up to big and tall; Physique is healthy and strong, and the body body is long, and chest leg flesh is flourishing; The muscle form has characteristics; Qingyuan City fiber crops chicken is a kind of native hen kind of China, and fat is flourishing between small, subcutaneous and flesh with it, the thin bone of skin is soft and famous, has become one of small-sized meat famous product chicken of China's live chickens outlet now.
Choose 10 of western Shandong cockfighting that are in growth curve flex point place (210 to 260 age in days), 8 female 2 public affairs, 19 of Qingyuan City fiber crops chickens, 13 female 6 public affairs, above-mentioned experiment is in full accord with the feeding and management condition of chicken.
The skeletal muscle fiber of difference quantitative test western Shandong cockfighting and Qingyuan City fiber crops chicken.Experimental technique is following:
1) sampling:
Western Shandong cockfighting and Qingyuan City fiber crops chicken stop raising 24 hours (potable water regular supply) back oral cavity bloodletting and butcher; Western Shandong cockfighting and Qingyuan City fiber crops chicken butchered after the bloodletting are immersed in the cold water; Fully drench feather, draining is placed on the operating table then, sprays oxydol in the fowl body; Cut off skin along carina; 1/3 place is reverse along muscle fibre on the musculus pectoralis superficialis of left side, gathers 2cm * 0.5cm * 0.5cm rectangular parallelepiped muscle masses, and immersing in liquid nitrogen is preserved.
2) preparation frozen section:
Start Leica CM1900 freezing microtome, biopsy chamber temperature-25 ℃ is set, freezing temperature is-20 ℃, and slice thickness is 10um, precooling 1h; Sample is taken out from liquid nitrogen, put into balance 0.5h in the Frozen Section Room; Take out sample; Be trimmed to the tissue slice of thick 0.5cm; On the freezing head of precooling, drip OCT frozen section embedding medium; Rapidly tissue is stained with, OCT frozen section embedding medium is smeared till covering fully in the edge of edge tissue clockwise, leaves standstill until OCT frozen section embedding medium all to become white; Freezing head is fixed on the freezing headstock together with organizing, and the screw of screwing puts down exhibition sheet plate; Slow rotary handle; Up to switching to tissue appearance,, glue with the microslide of handling rapidly and remove histotomy in case there is complete histotomy to occur; Mark is good, puts into the Glass carrier box of biopsy chamber precooling.
3) succinate dehydrogenase (SDH) dyeing:
1h before the dyeing puts into preheating in 37 ℃ of incubators with the dyeing liquor for preparing in advance; The component of dyeing liquor is following: 0.2M sodium succinate solution 100ml, 0.2M NaH
2PO
46.5ml, 0.2M Na
2HPO
443.5ml, 0.1% NBT (NBT) 200ml, deionized water 50ml; From Glass carrier box, take out the microslide that is stained with histotomy, under the room temperature, natural airing to section is turned white; Microslide is put in the dyeing liquor of preheating, hatched 40min for 37 ℃; After taking out microslide, slow flushing 30s in deionized water puts in 10% neutral formalin fixedly 10min; Section after fixing is slowly washed 30s with deionized water again, uses the ethanol of gradient concentration (75%, 85%, 95% and 100%) to dewater step by step then; Using volume ratio again is 2: 1-1: the section after 2 ethanol, the transparent dehydration of xylene mixed liquor transition; At last with the section of 100% xylene hyaline tissue, glycerin gelatine mounting.
4) observe and take pictures:
After leaving standstill 2h; Just putting microscope at Olympus BX51 research grade biology, eyepiece 10X, object lens 10X observe stained down; The photographic subjects zone; Select microscope to carry to take " Show Scale (show rulers) " in the software, at this moment length to occur be 500 microns scale in the picture lower right corner, preserves.
5) Computer Analysis picture:
The analyzing and processing of frozen section picture all adopts Image Pro-plus 5.02 version softwares to accomplish.
Image-Pro Plus (IPP) software is the famous image processing and analyzing software of Media Cybernetics company, supports IMAQ, strengthens, and demarcates; Color Image Processing, counting is measured, and analyzes; Image labeling, image data base, report form generator; The function expansion is carried out in grand record, VBA macroprogramming, and comprehensively Internet supports.Simultaneously, support to use C++, expansion such as Vb is the specific function to oneself using specially.Support various input sources, various picture formats are supported large-size images especially. peculiar fluorescence; Material analysis module, and microscope control module (being used for micrometron control) are applicable to biomedicine; Life science, the graphical analysis in a plurality of fields such as commercial Application and processing.
Image-Pro Plus 5.02 can run on
Windows 32-bit operating system:
2000 (service pack 4), and
XP Professional (service pack 1 reaches version later on).
According to software requirement, with Image-Pro Plus be installed in computer " IpWin5 " under the catalogue; " personal settings file " (mainly being scale setting and self-defined macros) be installed in " D: under the root directory.
Before the batch picture is handled; Choose form and be kept perfectly, dye preferably the muscle fibre picture as template picture, the scale size adjustment when gathering according to the scale of software self and picture then makes that the length of two scales representatives is consistent; Set the gray-scale value of IPP subsequently, make it to meet the requirements.Other picture all carries out the seizure of target fibers and the statistical study of correlation parameter with this standard.In the picture selected the inside, configure the parameter of mensuration---area (Area), mean diameter (Diameter-mean), average gray value (Density-mean), accumulation gray-scale value (IOD).In muscle bundle, select representative fiber of red muscle and white muscle fiber at random; Analyze its gray-scale value with IPP; Can determine the scope of the gray-scale value of a fiber of red muscle and white muscle fiber this moment, and the upper limit of lower limit and white muscle fiber of choosing the fiber of red muscle scope is as the evaluation boundary of three kinds of muscle fiber typeses, the muscle fiber types between two boundary values; Be judged to be intermediate fiber, as shown in Figure 1.
The concrete operations step is following:
1. open software
Double-click " Image-Pro Plus ", open and get into the interface, click the file button in the upper left corner, open the picture of a tensor muscle fiber clear-cut;
2. change OD value
In groupization dyeing, the discriminating between the muscle fiber types is to distinguish through the depth degree of muscle fibre dye levels is different, show among the IPP, and be exactly the difference of intensity (OD value).In IPP software, the intensity form of acquiescence, representative be gray-scale value, promptly color is worth more for a short time more deeply, the shallow more value of color is big more.This obviously is not inconsistent with target, and in fact the dye levels of measurement is an OD value, and it is dark more to dye, and OD value is big more.
Concrete operations are following: click " measure-＞calibration-＞intensity "; Then at window mid point " new " button; Tap " std optical density " again, can see that the straight line in the window has become reverse curve this moment, puts " system " button of the top again; All operations all are to operate according to unified standard after making, click " close " at last and close window.
3. correction scale
Scale in scale in the software and the actual photographed picture is often inconsistent, for unified yardstick between the two, need proofread and correct scale in the IPP the inside, makes that the yardstick of software and pictures taken is consistent.Concrete operations are: click " measure-＞calibration-＞spatial "; Demarcate in the menu at the scale that ejects; Click then " new ", " spatial cal 0 " under " name " of the top item locates to tap, and makes English character " 500um " into; Under " UNIT " item, insert " um ", beat and collude for " Convert when change unit " option of " um " below.Click " image " in " pixels/unit " frame subsequently; Eject " scaling " dialog box, show a scale in the scale picture upper left corner simultaneously, mouse is moved on on the whippletree of this scale; Tap left button; Cursor just becomes little hand immediately, drags the scale place of cursor to picture, the two ends that drag the software scale with left button make it with picture on the justify align of scale.Like photo is under 10 times of object lens, 10 times of eyepiece conditions, to take, and takes unit and fills in " 500 ", clicks " OK " and closes " scaling " window, gets back to " spatial calibration " window, clicks " close " and finishes.If it is not right that scale is done, can directly click " delete ";
4, the muscle fibre parameter is selected
As required, selected measurement parameter commonly used, wherein, parameter commonly used has " Area ", " Density (mean) ", " Diameter (mean) ", " IOD (Integrated Optical Density) ".Measuring myofibrillar purpose is to measure its apparent index, so select the former three parameter index.The concrete operations step: point " measure-＞count/size-＞measure-＞select measure ", click target component, selected parameter is clicked " OK ", gets back to " count/size " interface, accomplishes parameter and selects.
5, target area (AOI) selects
AOI is the most useful, a most important notion among the Image-Pro Plus, and this also is the key operation in the Image-Pro Plus use.Myofibrillar cross-sectional area is not desirable circle, but various irregular closed figures.In order to reproduce myofibrillar actual parameter more truly, in this method can by the top, interface " Irregular " instrument
accomplish.The concrete operations step is following: a tool bar can appear in
that click the top, interface; Click " Trace " instrument that tracks; Collude " Auto " on right side preceding beating, the value with " Speed " is adjusted into " 1 " (can revise the direction that tracks at any time at a slow speed) simultaneously.
The title of this instrument is Magic wand.Mouse is moved to myofibrillar inner membrance among the figure, and cursor has just become evil spirit rod, taps at a position left button, has just chosen this position and the similar zone of gray scale on every side thereof.Here the quantitative scope of " similar " is decided by the value of Range.Bigger range value is bigger tonal range, taps and can choose a sheet of zone.Less range value then is less tonal range.Multiple spot is several down on a zone, just can choose the whole border in this zone.Rearmost point is right button once, has just confirmed an erose selection area.The smooth value is used for the smooth region border.Generally do not need smoothly, so default value is zero.If choose a zone back also will on same pictures, select another zone again, can not directly click, click the Multiple AOI button on the toolbar earlier, click add again, subsequent another selection zone of just can arriving clicks.Select a Multiple AOI--add that goes back again behind what a new zone to confirm, continue again next, up to having chosen all AOI.Arbitrary place taps right button end selection operation on picture at last.Think to increase again the selection zone again if finish the back,, just can access tool bar and continue to increase the selection zone as long as tap " new AOI " button again.If the muscle fibre profile is not too clear, can with " Auto " preceding to scratching out, manual assisted Selection AOI.
6, self-defined grand
" grand " in fact only is the effective tool of a series of operative combination to an operation, can effectively objectively guarantee to judge the consistance between the plurality of pictures.The prerequisite of operation is, in all pending photos, selects one to have the form typical picture preferably that is kept perfectly, dyes, and carries out careful look and the analysis to measure of selecting.After obtaining satisfied result, just can carry out grand establishment according to this set operation formula.
Preparation before the establishment macro operation: after each item selection is accomplished in " count/size " window, comprise various parameters selections, " the save setting " that click in the file menu is saved in another disk partition, called after English name.Open " statistics " window, obtain measured value after, " DDE Option " window in point " file " menu is provided with measurement data and passes the rule toward Excel.In the DDEOption window, from top to bottom, collude " Append next date set to the bottom " preceding beating.After having set " DDE option ", do not close " statistics " window, open a blank " Excel " form.
Record macro: open a typical photo, open " count/size " window, also have empty " statistics " window, click " Macro-＞Record Macro ", just access the beginning window of record macro.
The step of record macro: " the file-load settings " of point " Count/size " window; Be selected in from the file that ejects and access " the count enactment document " preserved just now the menu; Click " Count ", counting, statistics can be presented in " Statistics " window at once; Click " file-DDEto Excel " in " Statistics " window, point " Shop recording " finishes record macro.For precisely, after each muscle fibre tracks later on, can use grand shortcut, determination data is kept in the file.
7. the differentiation of muscle fiber types
Select 3 ~ 5 typical pictures; Limit fiber of red muscle and white muscle fiber according to the groupization collection of illustrative plates; And use IPP software that its OD value is measured, subsequently with the OD value of white muscle fiber and fiber of red muscle according to from small to large series arrangement, the OD value minimum value of supposing white muscle fiber is I
W1, maximal value is I
W2The minimum value I of the OD value of fiber of red muscle
R1, maximal value is I
R2At this, think that artificially three kinds of myofibrillar OD values are respectively: white muscle fiber＜I
W2, intermediate fiber I
W2~ I
R1, fiber of red muscle＞I
R1, all myofibrillar data sinks General Logistics Department according to as above rule, compares, and analyzes muscle fiber types.
In this experiment, the OD value minimum value of white muscle fiber is that 0.1483 maximal value is 0.1821; The minimum value 0.2644 of the OD value of fiber of red muscle, maximal value is 0.4735.＜0.1821 be white muscle fiber, 0.1821 ~ 0.2644 be intermediate fiber,＞0.2644 be fiber of red muscle.
On histioid muscle fibre somatotype; Again skeletal muscle tissue is divided into three type: RMW (being mixed with the skeletal muscle fibre component type of fiber of red muscle, intermediate fiber and white muscle fiber), MW (being mixed with the skeletal muscle fibre component type of intermediate fiber and white muscle fiber), W (the skeletal muscle fibre component type that is made up of white muscle fiber fully), as shown in Figure 2.Further analyze the muscle fibre number that obtains in various myofibrillar average gray values, mean diameter, the muscle bundle, every type of myofibrillar number and shared ratio or the like.The determination data that the quantification of being gathered is described is used for the multiple comparative analysis of Duncan.
Two, the result of the skeletal muscle fiber of the cockfighting of quantitative test western Shandong and Qingyuan City fiber crops chicken
1) three types of muscle fibre distributions in different skeletal muscle tissues
Show that from section statining observation and two aspect analyses of average gray value mensuration SDH dyeing can be used for successfully the muscle fibre in the skeletal muscle being divided into three types.According to the situation of the contained fiber type of each research object, skeletal muscle is divided into three types again, that is: 1) the RMW type: contain fiber of red muscle, intermediate fiber and white muscle fiber; 2) MW type: contain intermediate fiber and white muscle fiber; 3) W type: only contain white muscle fiber.
(the number of elements of number of elements/test chicken of the chicken of number percent=each muscle fiber types of different musculus pectoralis superficialis types of organization of the number percent of different musculus pectoralis superficialis types of organization in western Shandong cockfighting and Qingyuan City fiber crops chicken; Number of elements/test chicken sum like RMW type number percent=RMW type chicken) analysis result shows; The fiber of red muscle of different proportion and the individuality of intermediate muscle fiber have all appearred containing in the musculus pectoralis superficialis in western Shandong cockfighting and Qingyuan City fiber crops chicken; The distribution proportion of individuality in western Shandong cockfighting and two kinds of Qingyuan City fiber crops chicken that has dissimilar musculus pectoralis superficialis seen Fig. 3; 30% individuality is arranged is the RMW type in the western Shandong cockfighting in detecting sample; And in Qingyuan City fiber crops chicken, having only 1 example RMW type musculus pectoralis superficialis to occur, it is individual that Qingyuan City fiber crops chicken near 95% is the W type that contains white muscle fiber merely.
The musculus pectoralis superficialis of the RMW type in western Shandong cockfighting and Qingyuan City fiber crops chicken has also shown evident difference.
Generally speaking; Though all have individual musculus pectoralis superficialis to be defined as the RMW type in Qingyuan City fiber crops chicken and the western Shandong cockfighting; But the two is on muscle fiber types ratio (the myofibrillar sum of muscle fiber types ratio=each myofibrillar number/RMW type chicken) still different (Fig. 4).The white muscle fiber ratio of Qingyuan City fiber crops chicken is high, and the ratio of intermediate fiber is low, and the fiber of red muscle proportional difference of the two is not obvious.The diameter of muscle fiber of western Shandong cockfighting is totally greater than Qingyuan City fiber crops chicken; No matter but for which chicken kind, always the fiber of red muscle diameter at same position is significantly less than the white muscle fiber diameter, the diameter of intermediate fiber is then between between the two; But more approach white muscle fiber, as shown in Figure 5.
2) analysis of growth traits and musculus pectoralis superficialis meat proterties
Two chicken kinds that this experiment is used have all been chosen the corresponding growth phase in growth curve flex point place in the musculus pectoralis superficialis time of drawing materials.Two kinds have difference slightly on body weight, but difference with insignificance.Three kinds of different breast muscles type individualities are compared, can find out that RMW type individual growth is slower, body weight is lighter, and MW type and W type whose body weight improve successively, and be as shown in Figure 6.The also similar body weight result of the long result of shin bone, the long remarkable shin bone long (P＜0.05) of shin bone that the RMW type is individual less than MW type individuality.
Shearing force be that waterpower (percentage of water loss) is the most frequently used index of reflection meat quality.From the angle of shearing force, the difference of western Shandong cockfighting and Qingyuan City fiber crops chicken is not remarkable, and the difference between the interior three types of musculus pectoralis superficialis of same kind is not remarkable, as shown in Figure 7 yet.The percentage of water loss aspect, the difference of two kinds is not remarkable, but the percentage of water loss of RMW type musculus pectoralis superficialis is starkly lower than other types, and as shown in Figure 8.The existence that fiber of red muscle is described has certain help to keeping muscle moisture, thereby can make the fresher and tenderer succulence of muscle.
Claims (5)
1. the method for a quantitative test animals skeletal muscle fiber comprises the steps:
1) gets the skeletal muscle of the animal of exsomatizing, process frozen section;
2) frozen section in the step 1) is carried out succinate dehydrogenase dyeing;
3) to step 2) section after the dyeing takes pictures, and preserves picture, with computing machine image processing and analyzing software picture carried out quantitative test then;
Succinate dehydrogenase dyeing in the said method comprises the steps:
A) with said frozen section in the succinate dehydrogenase dyeing liquor, hatch 40min for 37 ℃;
B) with the fixing 10min in 10% neutral formalin of the section after the dyeing in the step a);
C) ethanol of the section after fixing in the step b) with 75%, 85%, 95% and 100% gradient concentration is dewatered step by step;
D) using volume ratio is the section after the ethanol dehydration in 1: 2 to 2: 1 ethanol and the transparent step c) of xylene mixed liquor;
E) with the transparent section of handling through step d) of 100% xylene.
2. method according to claim 1 is characterized in that: said Computer Image Processing analysis software is Image Pro-plus 5.02 version softwares.
3. method according to claim 1 and 2 is characterized in that: said animal is a chicken.
4. claim 1 or 2 described methods are in the meat application in identification of poultry or fowl.
5. application according to claim 4 is characterized in that: said fowl is chicken.
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| CN103512885A (en) * | 2013-10-11 | 2014-01-15 | 江苏省家禽科学研究所 | Method for distinguishing muscle fiber type of duck skeletal muscle |
| CN109541239B (en) * | 2018-12-21 | 2021-09-24 | 云南农业大学 | Method for breeding Wuding chicken based on parathyroid hormone blood biochemical marker |
| CN109975294A (en) * | 2019-04-03 | 2019-07-05 | 天津市畜牧兽医研究所 | A kind of measuring method of musculature fiber sum |
| CN110910961B (en) * | 2019-12-04 | 2022-07-29 | 中国农业科学院农产品加工研究所 | Method for judging cooking processing suitability of raw meat based on muscle fiber type |
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| 朱道立等.大鼠骨骼肌纤维组织化学分型与肌球蛋白重链的功能.《解剖学报》.2007,第38卷(第1期),93-97. * |
| 陈佩林,朱道立.骨骼肌琥珀酸脱氢酶的组织化学方法探讨.《中国兽医杂志》.2003,第39卷(第2期),10-11. * |
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