CN115624012A - Methods of Promoting the In vitro Embryo Development of Chinese Soft-shelled Turtle - Google Patents
Methods of Promoting the In vitro Embryo Development of Chinese Soft-shelled Turtle Download PDFInfo
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- 238000000034 method Methods 0.000 title claims abstract description 15
- 230000013020 embryo development Effects 0.000 title claims abstract description 14
- 238000000338 in vitro Methods 0.000 title claims abstract description 8
- 230000001737 promoting effect Effects 0.000 title claims abstract description 7
- 241000736919 Pelodiscus sinensis Species 0.000 title claims description 9
- 235000013601 eggs Nutrition 0.000 claims abstract description 36
- 241001482311 Trionychidae Species 0.000 claims abstract description 26
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 claims abstract description 23
- 210000001161 mammalian embryo Anatomy 0.000 claims abstract description 13
- 230000018109 developmental process Effects 0.000 claims abstract description 12
- 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 24
- 239000011780 sodium chloride Substances 0.000 claims description 12
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 8
- 239000000243 solution Substances 0.000 claims description 6
- 230000008569 process Effects 0.000 abstract description 3
- 238000005507 spraying Methods 0.000 abstract description 3
- 230000000052 comparative effect Effects 0.000 description 17
- ONIBWKKTOPOVIA-BYPYZUCNSA-N L-Proline Chemical compound OC(=O)[C@@H]1CCCN1 ONIBWKKTOPOVIA-BYPYZUCNSA-N 0.000 description 15
- 102000008186 Collagen Human genes 0.000 description 6
- 108010035532 Collagen Proteins 0.000 description 6
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- 210000001519 tissue Anatomy 0.000 description 6
- 108010022452 Collagen Type I Proteins 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 230000012447 hatching Effects 0.000 description 5
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- 230000014509 gene expression Effects 0.000 description 4
- 241000272205 Columba livia Species 0.000 description 3
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- 150000001413 amino acids Chemical class 0.000 description 3
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- 238000013424 sirius red staining Methods 0.000 description 3
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- 230000015572 biosynthetic process Effects 0.000 description 2
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- 239000000835 fiber Substances 0.000 description 2
- 230000036039 immunity Effects 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 239000004475 Arginine Substances 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
- 102000002322 Egg Proteins Human genes 0.000 description 1
- 108010000912 Egg Proteins Proteins 0.000 description 1
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- 101000875067 Homo sapiens Collagen alpha-2(I) chain Proteins 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 108010029483 alpha 1 Chain Collagen Type I Proteins 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
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- 238000010586 diagram Methods 0.000 description 1
- 235000001434 dietary modification Nutrition 0.000 description 1
- 230000023011 digestive tract development Effects 0.000 description 1
- 230000008144 egg development Effects 0.000 description 1
- 230000007613 environmental 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
- 238000011534 incubation Methods 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
- 210000003205 muscle Anatomy 0.000 description 1
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- 210000004681 ovum Anatomy 0.000 description 1
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- 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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Abstract
一种促进中华鳖体外胚胎发育的方法，其特征在于选择中华鳖发育在第15期的胚蛋，在胚蛋中添加脯氨酸促进胚蛋发育。进一步，所述的脯氨酸注射到胚蛋的卵气室内。通过胚蛋给养脯氨酸可以干预胚胎发育过程，提高中华鳖品质性状，相对于大规模喷淋、饲喂等方式，更能精准的发挥作用。
A method for promoting in vitro embryonic development of soft-shelled soft-shelled turtle, which is characterized in that the development of embryonic eggs in the 15th stage of soft-shelled turtles is selected, and proline is added to the embryonic eggs to promote the development of embryon eggs. Further, the proline is injected into the air chamber of the embryonated egg. Supplying proline through embryo eggs can intervene in the embryonic development process and improve the quality traits of soft-shelled turtles. Compared with large-scale spraying and feeding, it can play a more precise role.
Description
技术领域technical field
本发明涉及一种调控中华鳖胚胎发育的方法。属于生物技术领域。The invention relates to a method for regulating the embryonic development of Chinese soft-shelled turtle. Belongs to the field of biotechnology.
背景技术Background technique
胚蛋给养是通过注射将外源营养物质补充到孵化期胚蛋中的给养方式，也称卵内给养。目前，胚蛋给养主要应用与家禽中，大量研究结果表明，卵内给养可改善雏鸡肠道健康，增加肌肉和肝脏糖原储备，促进器官发育，提高机体免疫力。其中维生素和氨基酸在胚蛋给养中改善生长发育的研究较多。如卵内注射精氨酸对家鸽(Columba livia)的孵化性能和生长发育具有影响；家鸽卵内注射组氨酸可以提高孵化率、缩短孵化周期、有利于肠道发育，但对后期生长性能几乎无影响。Embryo-egg feeding is a feeding method in which exogenous nutrients are supplemented into embryonated eggs at the hatching stage by injection, also known as in-ovo feeding. At present, embryo-egg feeding is mainly used in poultry. A large number of research results show that in-egg feeding can improve the intestinal health of chicks, increase muscle and liver glycogen reserves, promote organ development, and improve immunity. Among them, vitamins and amino acids have been studied in improving growth and development in embryonic egg nutrition. For example, intraovo injection of arginine has an effect on the hatching performance and growth of domestic pigeons (Columba livia); intraovo injection of histidine in domestic pigeons can increase the hatchability, shorten the incubation period, and be beneficial to intestinal development, but it has a negative effect on the later growth There is little impact on performance.
胚胎期是中华鳖优良性状和个体生长发育的重要时期，易受到外界环境的影响。胚胎发育期间的营养限制可能妨碍胚胎发育，目前常见的方法是于大规模喷淋、饲喂等方式，虽然在一定程度上也可以干预胚胎发育过程，促进胚蛋发育，但存在用料不够精准、时机把握不佳等因素，无法充分发挥功效。The embryonic period is an important period for excellent traits and individual growth and development of soft-shelled turtles, and is easily affected by the external environment. Nutrient restriction during embryonic development may hinder embryonic development. At present, the common methods are large-scale spraying and feeding. Although it can also intervene in the embryonic development process to a certain extent and promote embryonic egg development, the materials used are not accurate enough. , Poor timing and other factors, can not give full play to the effect.
发明内容Contents of the invention
本发明所要解决的技术问题是针对上述的技术现状而提供一种能精准的促进中华鳖体外胚胎发育的方法。The technical problem to be solved by the present invention is to provide a method that can accurately promote the in vitro embryonic development of soft-shelled turtles in view of the above-mentioned technical situation.
本发明解决上述技术问题所采用的技术方案为：一种促进中华鳖体外胚胎发育的方法，其特征在于选择中华鳖发育在第15期的胚蛋，在胚蛋中添加脯氨酸促进胚蛋发育。The technical scheme adopted by the present invention to solve the above-mentioned technical problems is: a method for promoting the in vitro embryonic development of Chinese soft-shelled turtle, which is characterized in that the embryonic egg that is developed in the 15th stage of Chinese soft-shelled turtle is selected, and proline is added to the embryonic egg to promote the development of the embryonic egg. development.
胚胎期中华鳖在第14期胚胎背甲尚未发育完全，第16期才开始出现裙边，选择第15期是考虑在该性状出现之前的敏感期或关键时期进行营养调控，更有利于裙边组织的发育。而如果操作过早，胚胎死亡率会增大。During the embryonic stage, the carapace of soft-shelled turtles is not yet fully developed at the 14th stage, and the skirt begins to appear at the 16th stage. The reason for choosing the 15th stage is to consider the nutritional regulation in the sensitive or critical period before the trait appears, which is more conducive to the skirt tissue development. And if the operation is too early, the embryonic mortality rate will increase.
所述的脯氨酸注射到胚蛋的卵气室内，不戳破卵膜，以免对胚蛋造成损伤，影响发育。The proline is injected into the air chamber of the embryo egg without puncturing the egg membrane, so as not to cause damage to the embryo egg and affect the development.
作为优选，其特征在于所述的脯氨酸溶解在氯化钠水溶液中形成注射液进行注射，注射量为2～10μL，脯氨酸浓度为10～30mg/mL，氯化钠百分比浓度为0～0.9％。Preferably, it is characterized in that the proline is dissolved in an aqueous sodium chloride solution to form an injection for injection, the injection volume is 2-10 μL, the proline concentration is 10-30 mg/mL, and the percentage concentration of sodium chloride is 0 ~0.9%.
作为最佳，所述注射量为5μL，脯氨酸浓度为20mg/mL，氯化钠百分比浓度为0.9％。Optimally, the injection volume is 5 μL, the proline concentration is 20 mg/mL, and the percentage concentration of sodium chloride is 0.9%.
作为优选，包括如下步骤：首先用75％乙醇对中华鳖卵表面进行消毒，接着用钻孔器在中华鳖卵气室位置开注射孔，通过微量进样器将脯氨酸的氯化钠溶液注射到卵气室内，注射完毕后用封口膜封住注射孔。注射孔不易太大，1mm左右比较合适。As preferably, comprise the following steps: first sterilize the surface of soft-shelled turtle eggs with 75% ethanol, then open the injection hole at the position of the air chamber of soft-shelled turtle eggs with a drill, and inject the sodium chloride solution of proline by a micro-injector Inject into the air chamber of the egg, and seal the injection hole with a parafilm after the injection. The injection hole should not be too large, about 1mm is more suitable.
与现有技术相比，本发明的优点在于：脯氨酸(Proline，Pro)是一种结构独特的亚氨基酸，被称为蛋白质的基本组成部分，是具有仲胺的唯一蛋白源氨基酸，有助于胶原蛋白的构建，对于维持生命、生长、摄食、营养利用、免疫、抵御环境和病原应激都是必需的，被归为是功能性氨基酸。中华鳖裙边富含胶原蛋白，而脯氨酸是构成胶原三螺旋结构的重要组成之一，因此采用脯氨酸可以促进胶原积累，得到更优品质的中华鳖个体。Compared with the prior art, the present invention has the advantages that: Proline (Proline, Pro) is a structurally unique imino acid, known as the basic component of protein, and is the only protein source amino acid with secondary amines. Contributes to the construction of collagen, is essential for sustaining life, growth, feeding, nutrient utilization, immunity, and defense against environmental and pathogenic stress, and is classified as a functional amino acid. The skirt of Chinese soft-shelled turtle is rich in collagen, and proline is one of the important components that constitute the triple helix structure of collagen. Therefore, the use of proline can promote collagen accumulation and obtain better-quality Chinese soft-shelled turtle individuals.
通过胚蛋给养脯氨酸可以干预胚胎发育过程，提高中华鳖品质性状，相对于大规模喷淋、饲喂等方式，更能精准的发挥作用。裙边是中华鳖最具特征性的营养组织，脯氨酸能够在胚胎期就可以促进裙边生长和发育，有利于中华鳖幼体出壳后形成宽厚肥大裙边等优良品质性状。Supplying proline through embryo eggs can intervene in the embryonic development process and improve the quality traits of soft-shelled turtles. Compared with large-scale spraying and feeding, it can play a more precise role. The skirt is the most characteristic vegetative tissue of Chinese soft-shelled turtle. Proline can promote the growth and development of the skirt at the embryonic stage, which is conducive to the formation of good quality traits such as wide, thick and plump skirt after the larvae hatch.
附图说明Description of drawings
图1为实施例第22期和对比例第22期背甲比值对比图。Fig. 1 is a comparison chart of the carapace ratio of the 22nd period of the embodiment and the 22nd period of the comparative example.
图2为实施例第22期和对比例第22期背甲指数对比图。Fig. 2 is a comparison chart of the carapace index of the 22nd period of the embodiment and the 22nd period of the comparative example.
图3为实施例第22期和对比例第22期胚胎重对比图。Figure 3 is a weight comparison diagram of the embryos at the 22nd stage of the embodiment and the 22nd stage of the comparative example.
图4为实施例第22期和对比例第22期胚胎长对比图。Fig. 4 is a comparison chart of embryo length at stage 22 of the embodiment and stage 22 of the comparative example.
图5为实施例第22期和对比例第22期裙边比值对比图。Fig. 5 is a comparison chart of the skirt ratio of the 22nd period of the embodiment and the 22nd period of the comparative example.
图6为实施例第22期和对比例第22期裙边指数对比图。Fig. 6 is a comparison chart of the skirt index of the 22nd period of the embodiment and the 22nd period of the comparative example.
图7为实施例第22期和对比例第22期中华鳖裙边组织COL1A1表达水平对比图。Fig. 7 is a comparison chart of the expression level of COL1A1 in soft-shelled turtle skirt tissue in the 22nd stage of the example and the 22nd stage of the comparative example.
图8为实施例第22期和对比例第22期中华鳖裙边组织COL1A2表达水平对比图。Fig. 8 is a comparison chart of the expression level of COL1A2 in soft-shelled turtle skirt tissue in the 22nd stage of the example and the 22nd stage of the comparative example.
图9为实施例第22期天狼星红染色显微照片。Fig. 9 is a photomicrograph of Sirius red staining in the 22nd phase of the embodiment.
图10对比例第22期天狼星红染色显微照片。Figure 10 is a photomicrograph of Sirius red staining in the 22nd phase of the comparative example.
图11为实施例第22期和对比例第22期天狼星红I型胶原百分占比对比图。Fig. 11 is a comparison chart of the percentage proportion of Sirius red type I collagen in the 22nd period of the embodiment and the 22nd period of the comparative example.
具体实施方式Detailed ways
以下结合附图实施例对本发明作进一步详细描述。The present invention will be further described in detail below in conjunction with the accompanying drawings and embodiments.
实施例，首先用75％乙醇对中华鳖卵(发育在第15期的胚蛋)表面进行消毒，接着用钻孔器在中华鳖卵气室位置开注射孔，通过微量进样器将脯氨酸的氯化钠溶液注射到卵气室内，注射完毕后用封口膜封住注射孔。注射量5μL，脯氨酸的浓度为20mg/mL，氯化钠百分比浓度为0.9％。实施例中选用12个中华鳖卵。Embodiment, at first 75% ethanol is used to sterilize the surface of soft-shelled turtle eggs (embryo eggs at the 15th stage of development), then use a drill to open injection holes at the air chamber position of soft-shelled turtle eggs, and inject proline The sodium chloride solution of the acid is injected into the egg cell, and the injection hole is sealed with a parafilm after the injection is completed. The injection volume is 5 μL, the concentration of proline is 20 mg/mL, and the percentage concentration of sodium chloride is 0.9%. Select 12 Chinese soft-shelled turtle eggs for use in the embodiment.
对比例，首先用75％乙醇对中华鳖卵(发育在第15期的胚蛋)表面进行消毒，接着用钻孔器在中华鳖卵气室位置开注射孔，通过微量进样器将脯氨酸的氯化钠溶液注射到卵气室内，注射完毕后用封口膜封住注射孔。注射量5μL氯化钠水溶液，氯化钠百分比浓度为0.9％。对比例中选用12个中华鳖卵。Comparative example, at first 75% ethanol is used to sterilize the surface of soft-shelled turtle eggs (embryonic eggs developed at the 15th stage), then use a drill to open an injection hole at the position of the air chamber of soft-shelled turtle eggs, and inject proline by a micro-injector. The sodium chloride solution of the acid is injected into the egg cell, and the injection hole is sealed with a parafilm after the injection is completed. The injection volume is 5 μL of sodium chloride aqueous solution, and the percentage concentration of sodium chloride is 0.9%. In the comparative example, 12 soft-shelled turtle eggs were selected.
表一为实施例和对比例中华鳖卵重量统计：Table 1 is the weight statistics of soft-shelled turtle ovum of embodiment and comparative example:
结果：result:
存活率：由试验结果可知，实施例中中华鳖卵第22期孵化率为51％，对比例中中华鳖卵第22期的孵化率为42％，同等条件下实施例中的孵化率显著高于正常组(P＜0.05)。Survival rate: As can be seen from the test results, the hatching rate of the 22nd phase of the soft-shelled turtle egg in the embodiment is 51%, and the hatching rate of the 22nd phase of the soft-shelled turtle egg in the comparative example is 42%, and the hatching rate in the embodiment under the same conditions is significantly high in the normal group (P<0.05).
对胚胎期中华鳖外部形态的影响：对实施例和对比例中胚胎在第22期进行取样，用游标卡尺对胚胎长、背甲长、背甲宽、裙边宽进行测量，用电子天平测定胚胎重，背甲重以及裙边重。试验结果结合图1～6所示，注射Pro后，中华鳖胚胎更重，背甲裙边发育更好，总的来说，胚胎生长发育优于正常组。The impact on the external morphology of the soft-shelled turtle in the embryonic period: the embryos in the examples and comparative examples were sampled at the 22nd stage, and the length of the embryo, the length of the carapace, the width of the carapace, and the width of the skirt were measured with a vernier caliper, and the embryo was measured with an electronic balance. Heavy, heavy carapace and heavy skirt. The test results are shown in Figures 1 to 6. After the injection of Pro, the embryos of soft-shelled turtles were heavier, and the carapace skirt developed better. In general, the embryo growth and development were better than those of the normal group.
对裙边组织I型胶原基因表达的影响：结合图7和图8所示，实施例中I型胶原基因表达量更高，有利于I型胶原的合成。Effects on gene expression of type I collagen in skirt tissue: as shown in Figure 7 and Figure 8, the expression level of type I collagen gene in the embodiment is higher, which is beneficial to the synthesis of type I collagen.
对裙边组织天狼星红染色的影响：结合图9～图11所示，对比例裙边组织折光性不是很强，胶原纤维呈现亮红色，实施例胶原纤维也呈现亮红色，折光性更加明显。并且I型胶原蛋白百分占比结果表明，实施例显著高于对比例(P＜0.01)。Effect on Sirius Red staining of the skirt tissue: As shown in Figures 9 to 11, the skirt tissue of the comparative example is not very refractive, and the collagen fibers are bright red, while the collagen fibers of the example are also bright red, and the refraction is more obvious. And the results of the percentage of type I collagen showed that the examples were significantly higher than the comparison examples (P<0.01).
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