Method for accurately determining poultry meat lignification and application thereof

Abstract

The present application belongs to the technical field of poultry genetic breeding and breeding, and particularly relates to a method for accurately determining poultry meat woodification and application. The present application determines and analyzes the histological morphological indexes of muscle fiber of a large population HE section, and screens the key index of intramuscular membrane thickness related to chicken wood meat. Further effectiveness verification is carried out, and the results show that the intramuscular membrane thickness exists significant differences in the normal group and the wood meat groups of different degrees, and the Pearson correlation with the chest muscle woodification quantitative index compression force is above 0.5. Generally, for 42-day-old white-feathered broilers, when the intramuscular membrane thickness of the chest muscle is more than 1.7 times of the apparent score of the normal group, the individual can be determined as wood meat. The intramuscular membrane thickness disclosed by the present application can be used as a key index for auxiliary judgment of chicken wood meat, and has important practical application value in reducing the incidence of wood meat and improving meat quality through genetic or nutritional means.

Description

Technical Field

[0001] This invention belongs to the field of poultry genetics, breeding and farming technology, and specifically relates to a method for accurately determining the lignification of poultry meat and its application. Background Technology

[0002] Chicken is the world's largest meat product, with global chicken production reaching 121 million tons, accounting for approximately 35.1% of the world's total meat production (2021, FAO). Broiler chickens have significant advantages such as low feed conversion ratio, fast growth rate, and high production efficiency, contributing 70% of the global chicken production and playing an important role in ensuring meat supply, food security, and even economic development and social stability.

[0003] The main performance advantages of broiler chickens are rapid growth rate, high meat yield, and high feed conversion ratio. However, with the extreme selection of meat production performance, the incidence of new inferior meat types (called heterogeneous meat), such as woody breast (WB) and white striping (WS), has been increasing year by year. Among them, woody breast is more common and has the most significant impact on meat quality. The scoring of woody breast is mainly based on the degree of hardening of the breast muscle and the size of the diffuse area, with an incidence rate of 6.6%-89%; and the incidence rate has been increasing year by year. Similar reports have also been made in poultry such as turkeys in recent years.

[0004] In conclusion, the current high incidence of heterogeneous meat such as woody meat is causing huge losses to the poultry farming industry. By using precise identification methods for woody meat, we can assist in the breeding of high-quality, fast-growing white-feathered broiler breeds and also help to alleviate its incidence through nutritional means. Summary of the Invention

[0005] This invention provides a method and application for accurate determination of lignification in poultry meat, which solves the defects of existing lignified meat detection technology, such as strong subjectivity (apparent scoring) and the influence of sample placement time (compression force) on the accuracy of detection results.

[0006] In a first aspect, the present invention provides the application of endothelial thickness in determining heterogeneous meat in poultry.

[0007] The process by which this invention discovered that endothelial membrane thickness is suitable as an indicator for identifying heterogeneous meat in poultry is as follows:

[0008] This invention involves collecting tissue samples from areas of high incidence of woody meat (generally the thickest part of the pectoral muscle) immediately after slaughter of broiler chickens in the target population, and fixing them in 4% paraformaldehyde fixative for 48 hours.

[0009] After fixation, pectoral muscle tissue was prepared using the standard HE sectioning procedure, which included dehydration, clearing, paraffin infiltration and embedding. The tissue was then cut into 5-8 μm thick sections using a microtome. After further mounting and dewaxing, the sections were stained in hematoxylin solution for 5 min, followed by dehydration and clearing. Finally, the sections were fixed with neutral resin to prepare HE sections.

[0010] HE sections were placed under an upright fluorescence microscope. Three 50× areas with good field of view were selected from each sample section, and the histological morphological parameters of myofibrils were measured using Image Pro Plus 6.0 software.

[0011] For the initial test, a small number of individual samples need to be tested to determine the differentiation criteria between woody and normal muscle tissue. This involves testing at least 20 woody and 20 normal samples from the target group based on their appearance scores, comparing the degree of change in endothelial thickness of the woody muscle tissue samples compared to the normal samples, and then using this standard to determine the individual woody muscle tissue in the target group.

[0012] More specifically, in the application of the endomysial thickness provided by the present invention to determine the foreign meat in poultry, more than 20 samples of the poultry to be tested are collected to measure the endomysial thickness of the muscle and obtain the average endomysial thickness of the poultry to be tested; with the endomysial thickness of normal meat as a control, if the average endomysial thickness of the poultry to be tested is more than 1.7 times higher than that of normal meat, then the poultry to be tested is determined to have foreign meat.

[0013] In the application of the endothelial thickness provided by this invention for determining heterogeneous meat in poultry, the heterogeneous meat is lignified meat.

[0014] In the application of endothelial thickness provided in this invention for determining heterogeneous poultry meat, the poultry is white-feathered broiler chicken.

[0015] Secondly, the present invention provides a method for detecting lignification in meat, which determines whether the meat has lignified by measuring the histological morphological indicators of the muscle fibers.

[0016] In the method for detecting lignification of meat provided by this invention, the histological morphological index of muscle fibers is the thickness of the myofascia.

[0017] In the method for detecting lignification of meat provided by the present invention, if the thickness of the endothelial membrane of the meat to be tested is more than 1.7 times higher than that of normal meat, the meat to be tested is determined to be lignified meat.

[0018] In the method for detecting lignification of meat provided by the present invention, the meat is poultry meat; preferably, the meat is chicken breast.

[0019] The beneficial effects of this invention are as follows:

[0020] This invention, through the determination of histological morphology indicators of muscle fibers of different strains with varying degrees of woodiness (no-mild-moderate-severe), has for the first time discovered that the thickness of the endothelial membrane differs significantly between the normal and abnormal groups in terms of appearance score, suggesting that the thickness of the endothelial membrane is a usable indicator for accurately determining woodiness.

[0021] This invention, through verification experiments on purebred roosters and commercial broiler chickens, further determined that when the average endothelial thickness is more than 1.7 times that of the average endothelial thickness of the normal apparent score group, an individual can be judged as a woody meat individual; and, 20 endothelial thicknesses were measured for each individual as representative values. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0023] Figure 1 These are HE-stained pathological sections of pectoral muscles from purebred broiler roosters in the normal group and the moderately woody group. The arrows indicate muscle fiber vacuoles, and the straight lines represent the perimysium and endomysium.

[0024] Figure 2 This is a statistical analysis of the morphological indicators of pectoral muscle fibers in purebred roosters of normal and moderately woody meat varieties. (0.001≤P<0.01:**; 0.0001≤P<0.001:***; P<0.0001:****).

[0025] Figure 3 These are HE-stained pathological sections of the pectoral muscles of Kobo commercial broiler chickens from the mild, moderate, and severe woody meat groups.

[0026] Figure 4 This is a statistical analysis of the myofascial index of the pectoral muscle in different groups of Kobo commercial broiler chickens (0.001≤P<0.01:**; 0.0001≤P<0.001:***; P<0.0001:****).

[0027] Figure 5 The results of statistical analysis of the compressive force of the pectoral muscles of different groups of purebred broiler roosters are shown in Figure A; the results of statistical analysis of the compressive force of the pectoral muscles of different groups of Kobo commercial broiler chickens are shown in Figure B.

[0028] Figure 6 The correlation and fitting equation between endothelial thickness and compressive force are (r = 0.515, P < 0.001).

[0029] Figure 7This is a statistical analysis of the number of different myofascial membranes in the pectoral muscles of purebred roosters in the normal group and the moderately woody meat group. Detailed Implementation

[0030] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0031] Example 1: Comparison of the histological morphology of pectoral muscle fibers in different groups of purebred broiler roosters

[0032] 1. Preparation of experimental samples:

[0033] 150 purebred broiler roosters were raised to 42 days of age and reached a weight of approximately 2500g. Blood was collected from the wing vein, and the roosters were euthanized by electric shock, bled, scalded, weighed, and eviscerated. The intact pectoralis major muscle was dissected and handed over to a specialist for heterogeneous meat scoring and grouping. A pectoralis major muscle tissue sample (2cm×1cm×0.2cm) parallel to the muscle fibers was taken and fixed in 4% paraformaldehyde fixative for 48 hours.

[0034] 2. Experimental methods:

[0035] (1) Grouping criteria for woody muscle: Woody muscle is scored in four levels based on the area, extent and hardness of the hardened pectoral muscle. Normal pectoral muscle (0 points): The entire pectoral muscle is soft and elastic, with a smooth and delicate surface and good flexibility; Mild woody muscle (1 point): The whole muscle is soft and flexible, the top of the pectoral muscle feels hard to the touch, and the rest of the muscle is elastic; Moderate woody muscle (2 points): The top to the middle of the pectoral muscle is hard, and the middle to the tail has a certain degree of elasticity; Severe woody muscle (3 points): The entire pectoral muscle is hard, stiff, and has ridge-like protrusions.

[0036] (2) Acquisition of endothelial parameters: Pectoral muscle tissue fixed for 48 hours was processed through dehydration, clearing, paraffin embedding, and embedding. The tissue was then sliced ​​into 5-8 μm thick sections using a microtome. After further mounting and dewaxing, the sections were stained in hematoxylin solution for 5 minutes. Dehydration and clearing were then performed, and finally, the sections were fixed with neutral resin and placed under an upright fluorescence microscope for image acquisition. The prepared paraffin sections of muscle were observed and photographed using an upright fluorescence microscope. Three 50× areas with good field of view were selected from each sample section. The acquired images were analyzed using Image Pro Plus 6.0 software (MediaCybernetics, Silver Spring, MSA).

[0037] (3) Collection and statistics of muscle fiber density: Two areas with uniform muscle fiber distribution and no perimysium were selected in each field of view for area measurement, and the number of muscle fibers in each area was counted, for a total of 5 muscle fiber density data points. Muscle fiber density was calculated as follows: Muscle fiber density (N / mm²) 2 = Number of muscle fibers / Muscle fiber area; the average value represents the individual's muscle fiber density. Data collection and statistics for the endomysium and perimysium: Muscle fibers uniformly distributed within muscle bundles were selected, and several fibers were chosen as centers. The distance between these fibers and their adjacent fibers was measured as the endomysium thickness. A total of 100 endomysium thickness data points were collected from three images, and the average value was calculated to represent the individual's endomysium thickness. Two adjacent muscle bundles were selected in each field of view, and the distance between the bundles was measured as the perimysium thickness. A total of 50 perimysium thickness data points were measured in three fields of view, and the average value was calculated to represent the individual's perimysium thickness.

[0038] 3. Experimental Results and Analysis:

[0039] The morphological characteristics of HE staining of terminal paternal pectoral muscle tissue in white-feathered broiler chickens are as follows: Figure 1 As shown, the woody flesh formation group exhibits muscle fiber dispersion, the appearance of round muscle fibers, and connective tissue hyperplasia.

[0040] The results of quantitative statistical analysis of pectoral muscle fiber morphology are as follows: Figure 2 As shown, the average muscle fiber diameter, area, and endothelial thickness in the moderately woody muscle group were 1.21, 1.43, and 1.78 times greater than those in the normal group, respectively, while the average muscle fiber density and perimysial thickness showed no significant differences. Specifically, the muscle fiber diameter in the moderately woody muscle group was 11.46 μm, the muscle fiber area was 151.88 μm², and the muscle fiber density was 6175.38 N / mm². 2 The perimysial membrane thickness was 10.27 μm, and the endomysial membrane thickness was 1.97 μm. In the normal group, the muscle fiber diameter was 9.48 μm, the muscle fiber area was 106.43 μm², and the muscle fiber density was 6807.69 N / mm². 2 The perimysium thickness is 12.27 μm, and the endomysium thickness is 1.11 μm.

[0041] Example 2: Comparison of the thickness of the endomysium of the pectoral muscle in different groups of commercial broiler chickens

[0042] 1. Preparation of experimental samples:

[0043] 120 Kobo commercial broiler chickens were raised to 42 days of age. After slaughter, the complete pectoralis major muscle was removed and the heterogeneous meat appearance of the right pectoralis major muscle was evaluated. A pectoralis major muscle tissue sample (2cm×1cm×0.2cm) parallel to the muscle fibers was taken and fixed in 4% paraformaldehyde fixative for 48h.

[0044] 2. Experimental methods:

[0045] The grouping criteria for woody flesh and the collection of endothelial indicators were the same as in Example 1.

[0046] 3. Experimental Results and Analysis:

[0047] The morphological characteristics of pectoral muscle tissue stained with HE are as follows: Figure 3 As shown, mild, moderate, and severe lignin formation all exhibited connective tissue hyperplasia. The results of quantitative statistical analysis of the pectoral muscle endothelial thickness are as follows: Figure 4 As shown, the endometrial thickness of mild, moderate, and severe woody muscle tissue was 2.81, 2.86, and 2.89 times that of the normal pectoral muscle endometrial thickness in purebred broiler roosters, respectively. Specifically, the average endometrial thickness was 3.12 μm in the mild woody muscle group, 3.18 μm in the moderate woody muscle group, and 3.21 μm in the severe woody muscle group.

[0048] Example 3 study determined that endothelial thickness is an important indicator related to lignin height.

[0049] 1. Preparation of experimental samples:

[0050] The samples are the same as those in Examples 1 and 2.

[0051] 2. Experimental methods:

[0052] (1) Grouping criteria for woody flesh: Same as in Example 1.

[0053] (2) Compression force measurement method: A texture analyzer was used, with a slender cylindrical probe of 6 mm in diameter. The experimental trigger force was set to 5 g, and the probe height was set to 55 mm (higher than the thickest part of the sample). The probe movement speed before compression was 10 mm / s; the probe movement speed during sample compression was 5 mm / s; the probe movement speed after compression was 10 mm / s; and the probe compression ratio was 20%. Three non-destructive compressions of 20% were performed on the top area of ​​the sample. The maximum force (CF) obtained after each compression was recorded, and the average of the three CF values ​​was used as the test result for a single sample. The compression force of all pectoral muscles was measured and calculated using the above method.

[0054] (3) Collection and calculation of endothelial indicators: Same as in Example 1.

[0055] (4) Pearson correlation analysis was performed on the compression force and wood pulp groups, and the regression equation was fitted using GraphPadPrism 8.

[0056] 3. Experimental Results and Analysis:

[0057] Pearson correlation analysis was performed on the compressive strength (CF) score and woody muscle score of purebred roosters and Cobb commercial broilers. The correlations between CF and woody muscle score reached 0.730 and 0.667, respectively, which are relatively high. Furthermore, previous literature has reported a correlation as high as 0.79 between CF and woody muscle score (Sun et al., 2018), making it a suitable indicator for quantitative evaluation. Simultaneously, the compressive force of each group was compared based on the pectoral muscle score, and statistical analysis was performed. The results are as follows: Figure 5 As shown. Pearson correlation analysis was performed on the individual endomysial thickness measured in both groups and the corresponding CF (cutaneous fat layer) of the pectoral muscle tissue, and the regression equation was fitted using GraphPad Prism 8. The results showed that the correlation between endomysial thickness and CF reached above 0.5 (e.g., ...). Figure 6 As shown in the figure, the woody flesh can be determined using the equation Y = 0.3373*X + 0.3185.

[0058] Statistical analysis was conducted on the endomysial thickness of pectoral muscle tissue in purebred broiler roosters and Cobb commercial broiler chickens with different degrees of woody texture. The results showed that in purebred broiler roosters, the average endomysial thickness in the normal group and the moderately woody group was 1.11 μm and 1.97 μm, respectively. Compared with the normal group, the endomysial thickness of moderately woody individuals was 1.78 times that of normal individuals. In Cobb commercial broiler chickens, the average endomysial thickness in the mild, moderate, and severe woody groups was 3.12 μm, 3.18 μm, and 3.21 μm, respectively. The gene expression levels of mild, moderate, and severe woody individuals were 2.81, 2.86, and 2.89 times that of normal purebred broiler roosters, respectively. Individuals with an average endomysial thickness greater than 1.7 times that of the normal group were considered to have woody texture.

[0059] Example 4: Standard setting of endothelial thickness in the test group and minimum sample size test

[0060] 1. Preparation of experimental samples:

[0061] Same as Example 1.

[0062] 2. Experimental methods:

[0063] (1) The grouping criteria for woody flesh and the collection of myometrial indicators are the same as in Example 1.

[0064] (2) Select muscle fibers that are evenly distributed within the muscle bundle, and choose several muscle fibers as centers. Measure the distance between these muscle fibers and their adjacent muscle fibers to obtain the endothelial thickness. Collect a total of 20, 40, 60, 80, and 100 endothelial thickness data points from three images, and calculate the average value to represent the individual's endothelial thickness.

[0065] 3. Experimental Results and Analysis:

[0066] The endothelial thickness of 20, 40, 60, 80, and 100 intramuscular tissue fragments in each individual of purebred broiler roosters in both the normal and moderately woody meat groups was measured and statistically analyzed (e.g., Figure 7 As shown in the figure, the results indicated that there were no significant differences between the normal group and the moderately woody meat group in terms of the number of measurements taken, and the means were similar. Specifically, the average endothelial thickness of the normal group (20, 40, 60, 80, and 100 measurements) was 0.98 μm, 1.00 μm, 1.04 μm, 1.02 μm, and 1.02 μm, respectively, while the average endothelial thickness of the moderately woody meat group (20, 40, 60, 80, and 100 measurements) was 1.96 μm, 1.94 μm, 1.98 μm, 1.99 μm, and 1.99 μm, respectively. The average endothelial thickness of the moderately woody meat in the 20, 40, 60, 80, and 100 endothelial thickness measurements were 2.01, 1.92, 1.89, 1.96, and 1.95 times that of the normal individual group of purebred broiler roosters, respectively. Screening was conducted based on the mean endothelial thickness of different groups and the ratio of the lignocelluloid group to the normal group. The results showed that 20 endothelial thickness measurements for each individual could be used as representative values.

[0067] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. The application of endomysial membrane thickness in determining lignification in broiler chickens, characterized in that, More than 20 samples of white-feathered broiler chickens were collected for the measurement of the endomysial membrane thickness of the muscle, and the average endomysial membrane thickness of the white-feathered broiler chickens was obtained. The endomysial membrane thickness of normal meat was used as a control. If the average endomysial membrane thickness of the white-feathered broiler chickens was more than 1.7 times higher than that of normal meat, it was determined that the white-feathered broiler chickens had lignification of meat.

2. A method for detecting lignification in meat, characterized in that, By measuring the thickness of the endomysium of meat, it is determined whether the meat has become lignified. If the thickness of the endomysium of the meat being tested is more than 1.7 times higher than that of normal meat, the meat being tested is determined to be lignified meat.

3. The method according to claim 2, characterized in that, The meat in question is poultry.

4. The method according to claim 3, characterized in that, The meat in question is chicken breast.

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