CN115624012A - Method for promoting in vitro embryo development of Chinese softshell turtle - Google Patents
Method for promoting in vitro embryo development of Chinese softshell turtle Download PDFInfo
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- CN115624012A CN115624012A CN202211335349.1A CN202211335349A CN115624012A CN 115624012 A CN115624012 A CN 115624012A CN 202211335349 A CN202211335349 A CN 202211335349A CN 115624012 A CN115624012 A CN 115624012A
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- 241001194850 Pelodiscus Species 0.000 title claims abstract description 27
- 238000000034 method Methods 0.000 title claims abstract description 18
- 230000013020 embryo development Effects 0.000 title claims abstract description 11
- 230000001737 promoting effect Effects 0.000 title claims abstract description 9
- 238000000338 in vitro Methods 0.000 title claims abstract description 8
- 235000013601 eggs Nutrition 0.000 claims abstract description 42
- 210000001161 mammalian embryo Anatomy 0.000 claims abstract description 25
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 claims abstract description 21
- 238000011161 development Methods 0.000 claims abstract description 13
- 230000018109 developmental process Effects 0.000 claims abstract description 13
- 230000008144 egg development Effects 0.000 claims abstract 2
- 238000002347 injection Methods 0.000 claims description 25
- 239000007924 injection Substances 0.000 claims description 25
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 22
- ONIBWKKTOPOVIA-BYPYZUCNSA-N L-Proline Chemical compound OC(=O)[C@@H]1CCCN1 ONIBWKKTOPOVIA-BYPYZUCNSA-N 0.000 claims description 18
- 239000011780 sodium chloride Substances 0.000 claims description 11
- 241000736919 Pelodiscus sinensis Species 0.000 claims description 10
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 8
- 238000007789 sealing Methods 0.000 claims description 6
- 239000000243 solution Substances 0.000 claims description 4
- 239000007864 aqueous solution Substances 0.000 claims description 3
- 230000000249 desinfective effect Effects 0.000 claims description 2
- 238000005553 drilling Methods 0.000 claims description 2
- 230000008569 process Effects 0.000 abstract description 3
- 238000005507 spraying Methods 0.000 abstract description 3
- 230000000052 comparative effect Effects 0.000 description 17
- 210000001519 tissue Anatomy 0.000 description 7
- 102000008186 Collagen Human genes 0.000 description 6
- 108010035532 Collagen Proteins 0.000 description 6
- 229920001436 collagen Polymers 0.000 description 6
- 230000012447 hatching Effects 0.000 description 6
- 108010022452 Collagen Type I Proteins 0.000 description 5
- 102000012422 Collagen Type I Human genes 0.000 description 4
- 230000014509 gene expression Effects 0.000 description 4
- 238000007796 conventional method Methods 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 210000002257 embryonic structure Anatomy 0.000 description 3
- 241000272205 Columba livia Species 0.000 description 2
- 241001482311 Trionychidae Species 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
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- 235000015097 nutrients Nutrition 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 101100328883 Arabidopsis thaliana COL1 gene Proteins 0.000 description 1
- 239000004475 Arginine Substances 0.000 description 1
- 241000282461 Canis lupus Species 0.000 description 1
- 102100033601 Collagen alpha-1(I) chain Human genes 0.000 description 1
- 102100036213 Collagen alpha-2(I) chain Human genes 0.000 description 1
- 241000272201 Columbiformes Species 0.000 description 1
- 229920002527 Glycogen Polymers 0.000 description 1
- 101000875067 Homo sapiens Collagen alpha-2(I) chain Proteins 0.000 description 1
- 241001330502 Stephania Species 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 108010029483 alpha 1 Chain Collagen Type I Proteins 0.000 description 1
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
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- 230000023011 digestive tract development Effects 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 229940096919 glycogen Drugs 0.000 description 1
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 1
- 125000001841 imino group Chemical group [H]N=* 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 230000007413 intestinal health Effects 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 238000001000 micrograph Methods 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 230000000050 nutritive effect Effects 0.000 description 1
- 230000005305 organ development Effects 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 244000144977 poultry Species 0.000 description 1
- 229930182852 proteinogenic amino acid Natural products 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 150000003335 secondary amines Chemical class 0.000 description 1
- 238000013424 sirius red staining Methods 0.000 description 1
- JTPDRYGRGBHNRZ-FHNDMYTFSA-M sodium;(2s)-pyrrolidine-2-carboxylic acid;chloride Chemical compound [Na+].[Cl-].OC(=O)[C@@H]1CCCN1 JTPDRYGRGBHNRZ-FHNDMYTFSA-M 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
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- 238000003786 synthesis reaction Methods 0.000 description 1
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- 229940088594 vitamin Drugs 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
Images
Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/02—Breeding vertebrates
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- Life Sciences & Earth Sciences (AREA)
- Environmental Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Zoology (AREA)
- Animal Husbandry (AREA)
- Biodiversity & Conservation Biology (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
A method for promoting the in vitro embryo development of Chinese softshell turtle is characterized by selecting embryo eggs of Chinese softshell turtle in the 15 th stage of development, and adding proline in the embryo eggs to promote the embryo egg development. Further, the proline is injected into the egg air chamber of the embryonated egg. The embryo development process can be intervened by feeding proline through embryo eggs, the quality character of the Chinese softshell turtle is improved, and the method can play a role more accurately compared with large-scale spraying, feeding and other modes.
Description
Technical Field
The invention relates to a method for regulating and controlling the embryonic development of Chinese softshell turtles. Belongs to the field of biotechnology.
Background
The feeding of the embryo eggs is a feeding mode of supplementing exogenous nutrient substances into the embryo eggs in the hatching period by injection, and is also called in-egg feeding. At present, the embryo egg feeding is mainly applied to poultry, and a large number of research results show that the in-ovo feeding can improve the intestinal health of chicks, increase the glycogen storage of muscles and liver, promote the organ development and improve the immunity of organisms. Among them, many studies have been made on the improvement of growth and development of vitamins and amino acids in the feeding of embryonated eggs. For example, in ovo injection of arginine has an influence on the hatching performance and growth development of domestic pigeon (Columba livia); the injection of histidine in pigeon eggs can improve the hatchability, shorten the hatching period and facilitate the intestinal development, but has almost no influence on the later growth performance.
The embryo stage is an important stage of the excellent characters and the individual growth and development of the Chinese softshell turtle and is easily influenced by the external environment. The conventional method is a large-scale spraying, feeding and other modes, although the conventional method can intervene in the embryonic development process to a certain extent and promote the development of embryonated eggs, the conventional method has the factors of insufficient precision of materials, poor timing and the like, and cannot fully exert the effect.
Disclosure of Invention
The invention aims to provide a method for accurately promoting the in vitro embryo development of the Chinese softshell turtle aiming at the technical current situation.
The technical scheme adopted by the invention for solving the technical problems is as follows: a method for promoting the in vitro embryo development of Chinese softshell turtle features that the embryo egg of Chinese softshell turtle at the 15 th stage is chosen and proline is added to said embryo egg to promote the development of embryo egg.
In the embryonic stage, the Chinese soft shell turtle has no fully developed embryonic dorsal concha in the 14 th stage, the skirt begins to appear in the 16 th stage, and the 15 th stage is selected to perform nutrition regulation in a sensitive stage or a key stage before the appearance of the character, so that the development of skirt tissues is facilitated. Whereas if the procedure is too early, embryo mortality will increase.
The proline is injected into the egg air chamber of the embryonated egg, and the egg membrane is not punctured, so that the embryonated egg is prevented from being damaged and the development is not influenced.
Preferably, the proline is dissolved in an aqueous solution of sodium chloride to form an injection, and the injection is performed, wherein the injection amount is 2 to 10 mu L, the proline concentration is 10 to 30mg/mL, and the sodium chloride percentage concentration is 0 to 0.9%.
Most preferably, the injection amount is 5. Mu.L, the proline concentration is 20mg/mL, and the sodium chloride percentage concentration is 0.9%.
Preferably, the method comprises the following steps: the method comprises the steps of disinfecting the surface of the Chinese softshell turtle egg by using 75% ethanol, opening an injection hole in the air chamber of the Chinese softshell turtle egg by using a drilling machine, injecting a proline sodium chloride solution into the egg air chamber by using a microsyringe, and sealing the injection hole by using a sealing film after injection. The injection hole is not easy to be too large, and is suitable for about 1 mm.
Compared with the prior art, the invention has the advantages that: proline (Pro) is a structurally unique imino acid, called the basic constituent of a protein, is the only proteinogenic amino acid with secondary amines, contributes to the construction of collagen, is essential for life support, growth, feeding, nutrient utilization, immunity, protection against environmental and pathogenic stresses, and is classified as a functional amino acid. The Chinese soft shell turtle skirt is rich in collagen, and proline is one of the important components forming the collagen triple-helix structure, so that the proline can promote collagen accumulation to obtain a Chinese soft shell turtle individual with better quality.
The embryo development process can be intervened by feeding proline through embryo eggs, the quality character of the Chinese softshell turtle is improved, and the method can play a role more accurately compared with large-scale spraying, feeding and other modes. The skirt is the most characteristic nutritive tissue of the Chinese softshell turtle, and proline can promote the growth and development of the skirt in the embryonic period, so that the Chinese softshell turtle is favorable for forming the excellent quality characters of wide and thick fat skirts and the like after the young Chinese softshell turtle is shelled.
Drawings
FIG. 1 is a comparison graph of the armor ratio at the 22 nd stage of the examples and the 22 nd stage of the comparative examples.
FIG. 2 is a comparison graph of the backsplash index at stage 22 of the example and at stage 22 of the comparative example.
FIG. 3 is a graph showing the comparison between the embryos at the 22 nd stage in the examples and the 22 nd stage in the comparative examples.
FIG. 4 is a comparison graph of the length of embryos at stage 22 in the examples and at stage 22 in the comparative examples.
FIG. 5 is a graph comparing the skirt ratio between the 22 nd phase of the example and the 22 nd phase of the comparative example.
FIG. 6 is a graph comparing the skirt indexes at stage 22 of the examples and at stage 22 of the comparative examples.
FIG. 7 is a graph showing the comparison of COL1A1 expression levels in the skirt tissue COL1 of Wastegia chinensis Benth in stage 22 in the examples and stage 22 in the comparative examples.
FIG. 8 is a graph showing the comparison of COL1A2 expression levels in the skirt tissue of Stephania sinensis at stage 22 in the examples and at stage 22 in the comparative examples.
FIG. 9 is a microphotograph of the red staining of sirius red at the 22 nd stage of the example.
FIG. 10 is a photomicrograph of a day wolf star red stain of comparative example No. 22.
FIG. 11 is a graph comparing the percent of type I collagen at phase 22 of the examples and at phase 22 of the comparative examples.
Detailed Description
The invention is described in further detail below with reference to the following examples of the drawings.
In the example, the surface of the soft-shelled turtle egg (embryo egg developed at the 15 th stage) was sterilized with 75% ethanol, an injection hole was opened at the air chamber position of the soft-shelled turtle egg by a drill, a sodium chloride solution of proline was injected into the egg air chamber by a microsyringe, and the injection hole was sealed with a sealing film after the injection. The injection amount is 5 mu L, the concentration of proline is 20mg/mL, and the percentage concentration of sodium chloride is 0.9%. In the embodiment, 12 Chinese softshell turtle eggs are selected.
In comparison, the surface of the Chinese soft-shelled turtle egg (embryo egg developed in the 15 th stage) was sterilized with 75% ethanol, an injection hole was drilled in the air chamber of the Chinese soft-shelled turtle egg using a drill, a sodium chloride solution of proline was injected into the air chamber of the Chinese soft-shelled turtle egg using a microsyringe, and the injection hole was sealed with a sealing film after the injection. The injection amount is 5 mu L of sodium chloride aqueous solution, and the percentage concentration of sodium chloride is 0.9%. In the comparative example, 12 Chinese soft-shelled turtle eggs were selected.
The first table shows the weight statistics of the Chinese softshell turtle eggs in the examples and comparative examples:
| serial number | Example weight g | Comparative example weight g |
| 1 | 2.3528 | 2.4497 |
| 2 | 2.3293 | 2.9630 |
| 3 | 2.7579 | 2.4859 |
| 4 | 2.6311 | 2.839 |
| 5 | 2.6773 | 2.3836 |
| 6 | 2.8366 | 3.4061 |
| 7 | 2.5693 | 2.6942 |
| 8 | 2.8936 | 2.3290 |
| 9 | 3.2660 | 2.6440 |
| 10 | 2.9804 | 2.6797 |
| 11 | 2.8077 | 3.2055 |
| 12 | 3.6806 | 3.6419 |
| Mean and standard deviation | 2.8152±0.3757 | 2.8101±0.4193 |
As a result:
survival rate: according to the test results, the hatching rate of the Chinese softshell turtle egg at the 22 nd stage in the example is 51%, the hatching rate of the Chinese softshell turtle egg at the 22 nd stage in the comparative example is 42%, and the hatching rate in the example is obviously higher than that in the normal group (P < 0.05) under the same conditions.
Influence on external morphology of the Chinese softshell turtle in the embryonic stage: the embryos of the examples and comparative examples were sampled at stage 22, and the embryo length, the length of the dorsal concha, the width of the dorsal concha and the width of the skirt were measured with vernier calipers, and the embryo weight, the dorsal concha weight and the skirt weight were measured with electronic balances. The test results are shown in the combined graphs of fig. 1-6, after Pro is injected, the Chinese soft-shelled turtle embryo is heavier, the dorsal concha skirt develops better, and the embryo growth and development are better than those of the normal group on the whole.
Effect on skirt tissue type I collagen gene expression: in the examples, as shown in FIGS. 7 and 8, the expression level of the type I collagen gene was higher, which was advantageous for the synthesis of type I collagen.
Effect on sirius red staining of skirt tissue: as shown in the combined images of FIGS. 9 to 11, the refractive index of the skirt tissue of the comparative example is not very strong, the collagen fibers are bright red, the collagen fibers of the example are also bright red, and the refractive index is more obvious. And the percentage of type I collagen results show that the examples are significantly higher than the comparative examples (P < 0.01).
Claims (4)
1. A method for promoting the in vitro embryo development of Chinese softshell turtle is characterized by selecting embryo eggs of Chinese softshell turtle in the 15 th stage of development, and adding proline in the embryo eggs to promote the embryo egg development.
2. The method for promoting the development of the embryo of trionyx sinensis as claimed in claim 1, wherein said proline is injected into the egg chamber of said embryo egg.
3. The method for promoting the in vitro embryonic development of the Chinese softshell turtle according to claim 2, wherein the proline is dissolved in the sodium chloride aqueous solution to form an injection, and the injection is carried out, wherein the injection amount is 2-10 μ L, the proline concentration is 10-30 mg/mL, and the sodium chloride percentage concentration is 0-0.9%.
4. The method for promoting the development of the embryo of the trionyx sinensis in vitro as claimed in claim 3, which comprises the following steps:
firstly, disinfecting the surface of the Chinese soft-shelled turtle egg with 75% ethanol, then opening an injection hole at the position of the air chamber of the Chinese soft-shelled turtle egg by using a drilling machine, injecting a sodium chloride solution of proline into the air chamber of the Chinese soft-shelled turtle egg by using a microsyringe, and sealing the injection hole by using a sealing film after the injection is finished.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211335349.1A CN115624012A (en) | 2022-12-19 | 2022-12-19 | Method for promoting in vitro embryo development of Chinese softshell turtle |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211335349.1A CN115624012A (en) | 2022-12-19 | 2022-12-19 | Method for promoting in vitro embryo development of Chinese softshell turtle |
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| CN115624012A true CN115624012A (en) | 2023-01-20 |
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| CN202211335349.1A Pending CN115624012A (en) | 2022-12-19 | 2022-12-19 | Method for promoting in vitro embryo development of Chinese softshell turtle |
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- 2022-12-19 CN CN202211335349.1A patent/CN115624012A/en active Pending
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