CN113080338A - Micropterus salmoides fatty liver model and construction method and application thereof - Google Patents
Micropterus salmoides fatty liver model and construction method and application thereof Download PDFInfo
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K50/00—Feeding-stuffs specially adapted for particular animals
- A23K50/80—Feeding-stuffs specially adapted for particular animals for aquatic animals, e.g. fish, crustaceans or molluscs
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K61/00—Culture of aquatic animals
- A01K61/10—Culture of aquatic animals of fish
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/20—Animal feeding-stuffs from material of animal origin
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/20—Animal feeding-stuffs from material of animal origin
- A23K10/22—Animal feeding-stuffs from material of animal origin from fish
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/30—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms
- A23K10/37—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms from waste material
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/142—Amino acids; Derivatives thereof
- A23K20/147—Polymeric derivatives, e.g. peptides or proteins
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/158—Fatty acids; Fats; Products containing oils or fats
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/163—Sugars; Polysaccharides
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/174—Vitamins
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/20—Inorganic substances, e.g. oligoelements
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/20—Inorganic substances, e.g. oligoelements
- A23K20/26—Compounds containing phosphorus
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
- Y02A40/81—Aquaculture, e.g. of fish
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
- Y02A40/81—Aquaculture, e.g. of fish
- Y02A40/818—Alternative feeds for fish, e.g. in aquacultures
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
- Y02P60/80—Food processing, e.g. use of renewable energies or variable speed drives in handling, conveying or stacking
- Y02P60/87—Re-use of by-products of food processing for fodder production
Abstract
The invention discloses a micropterus salmoides fatty liver model and a construction method and application thereof, wherein the fatty liver model is constructed by feeding a feed containing 10-20% of fat, 5-8% of saccharides and 0.1-0.3% of lipopolysaccharide. The invention finds that the high-fat high-sugar combined lipopolysaccharide synergistically induces the nutritive fatty liver model of the micropterus salmoides to be most similar to the natural pathogenesis, and the modeling method is simple, convenient and feasible, high in modeling rate, good in repeatability and strong in practicability, and can provide reliable materials for subsequent experimental research. In addition, the invention combines the related indexes of liver fat content and liver tissue morphology, comprehensively constructs the classification diagnosis method of the nutritional fatty liver of the micropterus salmoides, and can provide a relatively accurate and efficient judgment basis for the classification evaluation of the nutritional fatty liver of the micropterus salmoides.
Description
Technical Field
The invention belongs to the technical field of construction methods of model animal disease models, and particularly relates to a fatty liver model and a construction method and application thereof.
Background
Micropterus salmoides (Micropterus salmoides), also known as Micropterus salmoides, are one of the freshwater economic fish species successfully introduced and popularized and cultured in China, and are rapidly developed in recent years and are ascended to form the 'fifth major fish'. With the continuous expansion of the culture scale, the intensification degree is continuously improved, the production is increasingly pursued to achieve low cost and high yield, the fish fat metabolism disorder is caused by factors such as excessive feeding, too high energy or deterioration of feed, water quality deterioration and the like, the incidence rate of fatty liver disease is greatly improved, diseased fish grow slowly, the feed coefficient is increased, the anti-stress capability is reduced, the culture efficiency and the muscle quality are seriously influenced, the immunity and the anti-stress capability of the fish are also greatly reduced, particularly, the mass death of the cultured fish is easily caused in high-temperature seasons, huge loss is caused to the production, and the healthy sustainable development of the industry is seriously restricted. Since the micropterus salmoides are not only congenital 'fatty liver patients' (the liver resists the deficiency of fat factors) but also congenital 'diabetes patients' (the tolerance to saccharides is low and the utilization rate of starch is low), the diagnosis and judgment of the fatty liver occurrence degree can effectively guide the healthy culture production of the micropterus salmoides.
At present, the construction of a nutritional fatty liver model of fishes is mainly completed by feeding high-fat feed. And the micropterus salmoides can achieve the nutritional type fatty liver synergistic induction effect through high-fat high-sugar feed because of the special digestion habits of high-fat tolerance and high-sugar tolerance. At present, no relevant literature report on construction of a nutritional type fatty liver model of micropterus salmoides is found, and the simple feeding of high-fat high-sugar feed is relatively time-consuming, so that a simple and efficient fish fatty liver model construction method is urgently needed.
Disclosure of Invention
A first object of the invention is to provide a feed.
The second purpose of the invention is to provide the application of the feed in constructing a fatty liver model.
The third purpose of the invention is to provide a method for constructing a fatty liver model.
The fourth purpose of the invention is to provide a micropterus salmoides fatty liver grading evaluation method.
The technical scheme adopted by the invention is as follows:
in a first aspect of the invention, there is provided a feed comprising the following ingredients: 10-20% of fat, 5-8% of saccharide and 0.1-0.3% of lipopolysaccharide.
In some embodiments of the invention, the feed consists of the following components in parts by weight: 35-40 parts of fish meal, 10-15 parts of chicken meal, 5-10 parts of peeled soybean meal, 8-10 parts of corn protein powder, 5-10 parts of high gluten flour, 1-3 parts of cuttlefish paste, 0.5-1 part of salt, 0.5-1.5 parts of monocalcium phosphate, 4-6 parts of soybean oil, 2-10 parts of fish oil, 1-2 parts of soybean lecithin, 1-2 parts of vitamin premix, 1-2 parts of mineral premix and 0-0.3 part of lipopolysaccharide.
In some preferred embodiments of the invention, the feed consists of the following components in parts by weight: 38 parts of fish meal, 10 parts of chicken powder, 6 parts of peeled soybean meal, 10 parts of corn protein powder, 10 parts of high-gluten flour, 2 parts of cuttlefish paste, 0.5 part of salt, 1 part of monocalcium phosphate, 6 parts of soybean oil, 8 parts of fish oil, 1 part of soybean lecithin, 1.5 parts of vitamin premix, 1.5 parts of mineral premix and 0.2 part of lipopolysaccharide.
In a second aspect of the invention, there is provided the use of a feed according to the first aspect of the invention for the construction of a fatty liver model.
In some embodiments of the invention, the fatty liver model is specifically a micropterus salmoides fatty liver model.
In a third aspect of the invention, a method for constructing a fatty liver model of micropterus salmoides is provided, and the feed of the first aspect of the invention is fed to micropterus salmoides.
In some preferred embodiments of the invention, the feeding time is 6-16 weeks.
In some preferred embodiments of the invention, the feeding is performed for 8 weeks.
In some embodiments of the invention, the body mass of the micropterus salmoides is 8-50 g.
In some embodiments of the invention, the daily feeding amount is 1-5% of the body mass.
In a fourth aspect of the present invention, a micropterus salmoides fatty liver grading evaluation is provided, wherein the positive area rate and the liver fat content of the fatty liver model constructed by the method of the third aspect of the present invention are tested for grading evaluation.
In some preferred embodiments of the present invention, the liver is specifically tested for positive area rate and liver fat content.
In some embodiments of the invention, the criteria for the ranking is:
when the positive area rate is more than 3 percent, the liver fat content is more than 5 percent, and the liver is defined as fatty liver;
when the positive area rate is 3-8%, the liver fat content is 5-10%, and the mild fatty liver is defined;
when the positive area rate is 8-12%, the liver fat content is 10-14%, and the liver is defined as medium fatty liver;
when the positive area rate is greater than 12%, the liver fat content is greater than 14%, and the liver is defined as severe fatty liver.
The invention has the beneficial effects that:
aiming at the special digestion habits of high fat intolerance and high sugar intolerance of micropterus salmoides, lipopolysaccharide feeding is further increased on the basis of feeding high-fat high-sugar feed to promote liver metabolic damage, and further the rapid and efficient synergistic induction effect of nutritional fatty liver is achieved.
The method for inducing the nutritional type fatty liver model of the micropterus salmoides by the cooperation of high fat and high sugar and lipopolysaccharide is most similar to the natural pathogenesis, the modeling method is simple, convenient and feasible, high in modeling rate, good in repeatability and strong in practicability, the modeling time can be shortened by more than 4 weeks compared with that of the conventional high fat induction method, and reliable materials can be provided for subsequent experimental research. In addition, the invention combines the related indexes of liver fat content and liver tissue morphology, comprehensively constructs the classification diagnosis method of the nutritional fatty liver of the micropterus salmoides, and can provide a relatively accurate and efficient judgment basis for the classification evaluation of the nutritional fatty liver of the micropterus salmoides.
Drawings
FIG. 1 is a view of a liver tissue section in example 2.
FIG. 2 is a drawing showing the observation of a liver oil red O-stained tissue section in example 3.
Note: wherein A represents comparative example 1; b represents comparative example 2; c represents example 1; numbers represent weeks, microscope fold: 10X 20 times.
Detailed Description
The concept and technical effects of the present invention will be clearly and completely described below in conjunction with the embodiments to fully understand the objects, features and effects of the present invention. It is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments, and those skilled in the art can obtain other embodiments without inventive effort based on the embodiments of the present invention, and all embodiments are within the protection scope of the present invention.
Example 1
A feed comprises the following components (in parts by weight of air-dried substances): 38 parts of fish meal, 10 parts of chicken powder, 6 parts of peeled soybean meal, 10 parts of corn protein powder, 10 parts of high-gluten flour, 2 parts of cuttlefish paste, 0.5 part of salt, 1 part of monocalcium phosphate, 6 parts of soybean oil, 8 parts of fish oil, 1 part of soybean lecithin, 1.5 parts of vitamin premix, 1.5 parts of mineral premix and 0.2 part of lipopolysaccharide. The feed contains 19.97% of crude fat, 7% of saccharides and 0.2% of lipopolysaccharide.
Comparative example 1
A feed comprises the following components (in parts by weight of air-dried substances): 38 parts of fish meal, 10 parts of chicken powder, 6 parts of peeled soybean meal, 10 parts of corn protein powder, 7 parts of high-gluten flour, 2 parts of cuttlefish paste, 0.5 part of salt, 1 part of monocalcium phosphate, 6 parts of soybean oil, 3 parts of fish oil, 1 part of soybean lecithin, 1.5 parts of vitamin premix, 1.5 parts of mineral premix and 0 part of lipopolysaccharide. The feed contains 13.79% of crude fat, 5% of saccharides and 0% of lipopolysaccharide.
Comparative example 2
A feed comprises the following components (in parts by weight of air-dried substances): 38 parts of fish meal, 10 parts of chicken powder, 6 parts of peeled soybean meal, 10 parts of corn protein powder, 7 parts of high-gluten flour, 2 parts of cuttlefish paste, 0.5 part of salt, 1 part of monocalcium phosphate, 6 parts of soybean oil, 8 parts of fish oil, 1 part of soybean lecithin, 1.5 parts of vitamin premix, 1.5 parts of mineral premix and 0 part of lipopolysaccharide. The feed contains 19.97% of crude fat, 5% of saccharides and 0% of lipopolysaccharide.
Example 2
Constructing a nutritive fatty liver model of micropterus salmoides: juvenile Micropterus salmoides (8-50 g in body mass) are selected and fed with the feeds of example 1 and comparative examples 1-2 for 8 weeks respectively. Samples were taken at 1, 2, 4, 6, and 8 weeks after the start of the experiment. After the sampled fish is anesthetized by MS-222 of 100mg/L, the liver is quickly taken and fixed by 4% paraformaldehyde for observation of HE pathological tissue sections.
Judgment criteria for successful modeling:
the development status of fatty liver was judged by Image Pro-Plus 6.0 analysis from the liver cell (nucleus) morphology, vacuolation and steatosis degree scoring using the non-alcoholic fatty liver model scoring system (NAS) of Qing fish. When the average score of the three items reaches 2 minutes, the success construction of the micropterus salmoides high-fat nutritional type fatty liver model can be judged, wherein the score standard is shown in table 1, the dyeing result of the dry tissue section is shown in table 1, and the statistical result is shown in table 2.
TABLE 1 non-alcoholic fatty liver model scoring System (NAS) scoring criteria
TABLE 2 NAS System Scoring statistics for each Experimental group
The results show that under the experimental conditions, although the feeds of the comparative example 2 and the example 1 can successfully construct the nutritional type fatty liver model, the example 1 is obviously faster than the comparative example 2, wherein the feed of the example 1 can successfully construct the nutritional type fatty liver model at the 4 th week, and the feed of the comparative example 2 can successfully construct the nutritional type fatty liver model at the 6 th week.
Example 3
Nutritional fatty liver grading diagnosis method for micropterus salmoides
Juvenile Micropterus salmoides (8-50 g in body mass) are selected and fed with the feeds of example 1 and comparative examples 1-2 for 16 weeks respectively. Sampling at 0, 1, 2, 4, 6, 8, 10 and 16 weeks after the experiment is started, anesthetizing the fish sample by MS-222 with the concentration of 100mg/L, taking blood from the tail vein of a sterile syringe, standing for 2h at 4 ℃, centrifuging (1000g, 4 ℃ and 10min) after blood is layered, collecting serum in a 2mL centrifuge tube, and storing at-20 ℃ for serum biochemical index analysis. Thereafter, the liver was dissected and separated rapidly, and a part was frozen in liquid nitrogen for fat content measurement of the liver, and a part was fixed with 4% paraformaldehyde for fat content analysis by oil red O stained section, and the measurement results are shown in table 3. The results of oil red O staining are shown in FIG. 2, and the positive area rate of the liver was further analyzed by the results of oil red O staining, and the results are shown in Table 4.
TABLE 3 liver fat content changes of micropterus salmoides in each group
The difference in the superscript letters at the same sample points indicates significant difference (P < 0.05).
As can be seen from table 3. From week 4, significant differences in liver fat content occurred in each experimental group, and with increased levels of feed fat and carbohydrates and increased levels of fatty liver, the liver fat accumulation content increased (P < 0.05).
As can be seen in fig. 2, oil red O staining indicates the presence of red lipid droplets within the cells, where hepatocytes are stained blue as nuclei and orange-red as fat droplets. From week 4 on, the example 1 group began to develop a distinct orange-colored lipid droplet; comparative example 2 group developed a significant drop of fat starting from week 6; the group of comparative example 1 had no significant lipid droplets appeared until week 10. By week 16, three groups showed significant accumulation of orange-red lipid droplets, and the color gradually deepened with increasing feed levels and fatty liver levels.
TABLE 4 hepatocyte Positive area Rate (%) analysis
The difference in the superscript letters at the same sample points indicates significant difference (P < 0.05).
The results show that the liver positive area rate of different experimental groups is significantly different from week 4, and the positive area rate is increased with the increase of the feed fatty sugar level and the increase of the fatty liver degree (P < 0.05).
And (3) integrating the experimental results to construct a nutritional fatty liver diagnosis and grading evaluation standard:
(1) when the positive area rate is more than 3 percent, the liver fat content is more than 5 percent, and the liver is defined as fatty liver;
(2) when the positive area rate is 3-8%, the liver fat content is 5-10%, the liver is mild fatty liver;
(3) when the positive area rate is 8-12%, the liver fat content is 10-14%, and the liver is moderate fatty liver;
(4) when the positive area rate is greater than 12%, the liver fat content is greater than 14%, and the liver is defined as severe fatty liver.
The present invention is not limited to the above embodiments, and various changes can be made without departing from the spirit of the present invention within the knowledge of those skilled in the art. Furthermore, the embodiments of the present invention and the features of the embodiments may be combined with each other without conflict.
Claims (10)
1. A feed comprising the following ingredients: 10-20% of fat, 5-8% of saccharide and 0.1-0.3% of lipopolysaccharide.
2. The feed of claim 1, which consists of the following components in parts by weight: 35-40 parts of fish meal, 10-15 parts of chicken meal, 5-10 parts of peeled soybean meal, 8-10 parts of corn protein powder, 5-10 parts of high gluten flour, 1-3 parts of cuttlefish paste, 0.5-1 part of salt, 0.5-1.5 parts of monocalcium phosphate, 4-6 parts of soybean oil, 2-10 parts of fish oil, 1-2 parts of soybean lecithin, 1-2 parts of vitamin premix, 1-2 parts of mineral premix and 0-0.3 part of lipopolysaccharide.
3. Use of the feed of any one of claims 1-2 for constructing a fatty liver model.
4. Use according to claim 3, wherein the fatty liver model is in particular a fish fatty liver model, preferably micropterus salmoides.
5. A method for constructing a fatty liver model of micropterus salmoides, wherein the micropterus salmoides are fed with the feed as claimed in any one of claims 1-2.
6. The method according to claim 5, wherein the feeding time is 6 to 16 weeks.
7. The method according to claim 5, wherein the micropterus salmoides are juvenile fish having a body mass of 8-50 g.
8. The method according to claim 7, wherein the daily feeding amount is 1-5% by mass.
9. A method for grading fatty liver of Micropterus salmoides, which is used for grading and evaluating the positive area rate and the fat content of liver of a fatty liver model constructed by the construction method of any one of claims 5 to 8.
10. The rating method of claim 9, wherein the criteria for the rating are:
when the positive area rate is more than 3 percent, the liver fat content is more than 5 percent, and the liver is defined as fatty liver;
when the positive area rate is 3-8%, the liver fat content is 5-10%, and the mild fatty liver is defined;
when the positive area rate is 8-12%, the liver fat content is 10-14%, and the liver is defined as medium fatty liver;
when the positive area rate is greater than 12%, the liver fat content is greater than 14%, and the liver is defined as severe fatty liver.
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