CN114794082B - Antifreeze protein-containing diluent for improving preservation quality of chicken essence and preparation method thereof - Google Patents

Antifreeze protein-containing diluent for improving preservation quality of chicken essence and preparation method thereof Download PDF

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CN114794082B
CN114794082B CN202210396857.4A CN202210396857A CN114794082B CN 114794082 B CN114794082 B CN 114794082B CN 202210396857 A CN202210396857 A CN 202210396857A CN 114794082 B CN114794082 B CN 114794082B
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chicken
pine needle
tris
diluent
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CN114794082A (en
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周迪
蒋桂荣
杨蓉
王燕
赵忠海
唐明艳
王府
刘虓
任丽群
袁勇
谭晓山
敖叶
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Guizhou Livestock And Poultry Germplasm Determination Center
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02—Preservation of living parts
    • A01N1/0205—Chemical aspects
    • A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
    • A01N1/0221—Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02—Preservation of living parts
    • A01N1/0205—Chemical aspects
    • A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
    • A01N1/0226—Physiologically active agents, i.e. substances affecting physiological processes of cells and tissue to be preserved, e.g. anti-oxidants or nutrients

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Abstract

The invention provides a chicken semen cryopreservation diluent which is characterized by comprising the following components: pine needle antifreeze polypeptide, fructose, trehalose, potassium citrate monohydrate, magnesium acetate tetrahydrate, sodium glutamate, tris (Tris (hydroxymethyl aminomethane), reduced glutathione, oxidized glutathione and Dimethylacetamide (DMA). The chicken semen cryopreservation diluent is a chicken semen cryopreservation diluent which has the advantages of easily obtained raw materials, convenience in preparation, fertilization rate of more than 80% and higher DNA integrity. Compared with the related data of the fresh chicken essence, the DNA integrity is close to that of the fresh essence, the sperm motility and the hatching rate are slightly lower than those of the fresh essence, and the method has wide application prospect.

Description

Antifreeze protein-containing diluent for improving preservation quality of chicken essence and preparation method thereof
Technical Field
The invention relates to the field of biology, and relates to a diluent containing antifreeze protein and capable of improving the storage quality of chicken essence, a preparation method of the diluent, and further relates to application of the diluent in the frozen storage of the chicken essence.
Background
Semen cryopreservation is a great innovation of artificial insemination technology. The method solves the problem of long-term semen preservation, ensures that the semen is not limited by time, regions and the life of breeding stock, can fully utilize the genetic performance of an optimal stage of fine breeds, exerts and improves the utilization rate of the fine breeds of cocks to the utmost extent, greatly improves the genetic progress, and accelerates the breeding and the pace improvement of the breeds. Meanwhile, the descendant determination of the excellent breeder cock is carried out in a short period, the excellent characteristics of a certain breed or an individual cock are reserved and recovered, and the important significance and the application value are realized in aspects of blood system updating, introduction, production cost reduction, breed resource protection and the like.
For over 50 years, researchers at home and abroad and researchers of animal reproduction technology have conducted a great deal of research on techniques of semen cryopreservation and artificial insemination of livestock and poultry. Particularly, after Polge and the like in England of 1949 discover that glycerol (glycerol) has an anti-freezing protection effect on bovine sperms, the technical research and application are rapidly developed, and the application effect on dairy cattle is not obviously different from that of fresh sperms. Therefore, livestock workers at home and abroad have successively developed the research on the semen cryopreservation on the livestock and poultry of such species as cattle, sheep, horses, pigs, chickens, geese, ducks, turkeys and the like, and have made great progress and remarkable effect. A great deal of research has been conducted on poultry (geese, chickens, ducks, turkeys, etc.) mainly from the following points: at present, the chicken essence cryoprotectants mainly comprise glycerol, DMSO (dimethyl sulfoxide), DMA (N, N-dimethylacetamide), EG (ethylene glycol) and the like. On the other hand, however, it has been found that these chemical components also have a certain toxic effect on sperm (i.e., an anti-fertilization effect). Especially, the previous experiments of the invention show that glycerol, DMSO, DMA and EG all generate more remarkable toxic effects (P < 0.05) on the survival of sperms, wherein the toxicity of DMA is the minimum, and ethylene glycol and DMSO are the second. Therefore, screening out the high-efficiency, non-toxic and excellent cryoprotectant is one of the important links of the semen freezing of the breeding cock. In addition, semen cryopreservation not only relates to a preservation solution, but also relates to a specific freezing step and a specific thawing step, which directly influence the final fertilization effect.
The chicken semen freezing technology is an important means for preserving the germ plasm resources of poultry, and the semen freezing reagent is an important factor influencing the freezing preservation effect. The frozen semen reagent and the freezing operation method are mutually influenced, many frozen semen reagents which are simple and convenient to prepare are poor in effect, reagents with good effects are complex to prepare, and special equipment is needed for testing and adjusting.
The DNA integrity is an important semen quality index and is closely related to the fertilization rate, the aberration rate and the like. However, several methods with better effect do not provide semen quality indexes such as DNA integrity.
Although there are some reagents or methods in the prior art which are specially used for preserving chicken essence, there are some problems more or less, such as: CN104094926 discloses a chicken semen diluent, which comprises 7.0-9.0 g/L of fructose, 4.0-6.0 g/L of sodium glutamate, 11.0-14.0 g/L of dipotassium hydrogen phosphate, 0.5-0.7 g/L, TES (N-trimethylol methyl-2-aminoethanesulfonic acid) 3.0-5.0 g/L of potassium citrate, 3.0-6.0 g/L of sodium acetate trihydrate, 0.2-0.4 g/L of anhydrous magnesium chloride and 0.05-0.1 g/L of lycopene.
Chinese patent CN86101836 discloses a chicken semen diluent which contains glucose, sodium citrate, potassium dihydrogen phosphate, disodium hydrogen phosphate and distilled water. The diluted solution is chicken semen diluted by 1:2 and 1:1, the chicken semen is preserved for 7 and 24 hours at 5 ℃ and then inseminated, the fertilization rates of the eggs are 94.7 percent and 92.2 percent respectively, and the diluted chicken semen is suitable for inseminating immediately after dilution and also suitable for short-term preservation and inseminating after short-distance transportation. However, the patent does not solve the technical problem of long-term preservation of chicken essence, the problem that the transportation and insemination work of chicken essence cannot be busy fundamentally after 7-hour preservation, and the preservation time is required to be longer in practical production and application.
CN101543209A discloses a powder for chicken semen dilution and preservation, a preparation method and application thereof. The powder for diluting and preserving the chicken essence comprises 20 to 30 weight parts of glucose, 1.5 to 2.7 weight parts of sodium citrate, 35 to 45 weight parts of sodium glutamate and 55 to 75 weight parts of K 2 HPO 4 ·3H 2 O, 1 to 5 parts by weight of K 2 HPO 4 15-30 parts of sodium acetate trihydrate, 5-15 parts of NaCl and 1-9 parts of Tris by weight, and obtaining a diluent after adjusting the pH value by using distilled water to dissolveOptionally, a bactericide may be added. However, this diluent can be stored only in a low-temperature (4 ℃) environment, and cannot be stored frozen for a long period of time, and it is difficult to maintain its physiological activity after storage for a long period of time.
CN111406738A discloses a chicken essence diluent for artificial oviduct insemination and a preparation method thereof, wherein the chicken essence diluent comprises potassium citrate, fructose, sodium glutamate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium chloride hexahydrate, TES, sodium acetate and deionized water. However, the diluent can only be used for preserving the cock sperms at normal temperature, and is beneficial to prolonging the artificial insemination time of the cock and the utilization rate of the cock sperms. And does not provide long-term storage in a freezing environment such as liquid nitrogen.
Disclosure of Invention
In order to solve the technical problems, the invention aims to: provides a chicken semen freezing and storing diluent which has easily obtained raw materials, convenient preparation, fertilization rate of more than 80 percent and higher DNA integrity.
The chicken semen cryopreservation diluent comprises: pine needle antifreeze polypeptide, fructose, trehalose, potassium citrate monohydrate, magnesium acetate tetrahydrate, sodium glutamate, tris (Tris (hydroxymethyl aminomethane), reduced glutathione, oxidized glutathione and Dimethylacetamide (DMA);
more preferably, the ratio of the components is (1000 ml): 1-5ml of pine needle antifreeze polypeptide (1 mg/ml, dissolved in 001M PBS buffer), 5-10g of fructose, 8-20g of trehalose, 1-3g of potassium citrate monohydrate, 4-6g of magnesium acetate tetrahydrate, 11-19g of sodium glutamate, 8-13g of Tris (Tris (hydroxymethyl aminomethane), 0.1-0.5g of reduced glutathione, 0.05-0.1g of oxidized glutathione and 80-120 ml of DMA, and the balance of double distilled water;
the amino acid sequence of the pine needle antifreeze polypeptide is shown in SEQ ID NO.1 (Ser-Ala-Arg-Glu-Arg-Thr-Cys-Thr-Ser-Ser-Thr-Asn-Ser-Lys-Ala-Cys-Ala-Lys-Tyr-Thr-Pro-Thr-Asp-Thr-Cys-Thr), the polypeptide has a high-order structure of a beta-fold structure, and has a thermal hysteresis value of 0.6 ℃ in a 1mg/ml aqueous solution. Such polypeptides are preferred anti-freeze polypeptides at lower concentrations, i.e. with high thermal hysteresis values;
further, the invention provides an application of the chicken semen cryopreservation diluent, and the application is to prepare a chicken semen cryopreservation kit.
The invention further provides a method for freezing and preserving chicken semen by using the chicken semen freezing and preserving diluent, which comprises the following steps:
dissolving 5 to 10g of fructose, 8 to 20g of trehalose, 1 to 3g of potassium citrate monohydrate, 4 to 6g of magnesium acetate tetrahydrate, 11 to 19g of sodium glutamate, 8 to 13g of Tris (Tris (hydroxymethyl aminomethane), 0.1 to 0.5g of reduced glutathione and 0.05 to 0.1g of oxidized glutathione in 1000ml of water, fully dissolving, then adding 1 to 5ml of pine needle antifreeze polypeptide (1 mg/ml, dissolved in 001M PBS buffer), and fully dissolving and uniformly mixing to obtain a solution I; 660ml of the solution I is taken and added with 80 to 120ml of DMA to obtain a solution II, and the solution I, II is filtered and sterilized by a 0.22um filter and then subpackaged in a 2ml centrifuge tube for storage.
When in use, the solution I is preheated at 37 ℃, is fully and uniformly mixed and is subjected to isothermal dilution with original essence, and the original essence: liquid I =1:2; wrapping with gauze, placing in a refrigerator, slowly cooling to 4 deg.C within 1h, adding solution II for 1:1 dilution, placing in 0.25ml thin tube, freezing with a freezer, cooling from 5 deg.C to-35 deg.C at 7 deg.C/min, and then reducing to-140 deg.C at-60 deg.C/min.
Further, the invention provides a thawing method of frozen chicken semen, which comprises the following steps: thawing in water bath at 50 deg.C for 8s.
Advantageous effects
The invention has simple and easily obtained components, has high permeability, is beneficial to properly reducing intracellular moisture and reducing ice crystal damage; the pine needle antifreeze polypeptide is found in earlier research to ensure the stability of semen, has good buffering effect in the processes of freezing and unfreezing the semen and reduces the damage of ice crystals generated in freezing and refrigeration to the sperm; the phosphate has strong buffering capacity, so that the pH value is kept between 6.8 and 7.2, potassium ions can be provided, and the ionic balance is maintained; fructose is a saccharide which can be directly utilized by sperms, can provide energy for the sperms, and the mixed saccharide has a better protective effect on sperm membranes; and contains sodium glutamate which is very beneficial to sperm preservation; glutathione can reduce oxidative damage; the antifreeze protective agent DMA has the characteristic of low toxicity, and the antifreeze agent is not removed centrifugally during insemination.
Simple operation can use at large-scale not enough experimental condition's plant, does not need detecting instrument such as extra PH meter, potentiometre, osmometer, as long as guarantee according to the gauge of material, potassium ion: sodium ion =1 to 6, sodium glutamate: tris =1 = 0.9 to 1.0, glutathione reduced form: oxidized form =4 to 8, the frozen semen diluent is prepared according to the formula, so that the sodium potassium balance, osmotic pressure, pH and redox potential are in proper ranges, special instruments are not needed for detection, water and strong acid and alkali are not needed for adjustment, the whole process is extremely convenient to operate, and extra detection and quality monitoring are not needed.
Drawings
FIG. 1 is a photograph of a chicken sperm showing DNA integrity test in stages 0 and 1-4 showing progressively more damaged sperm;
FIG. 2 is a diagram showing a movement trace of a frozen chicken sperm and a sperm image A; b a less viable sperm track; and C, sperm track with better activity.
Detailed Description
Terms used in the present invention have generally meanings as commonly understood by one of ordinary skill in the art, unless otherwise specified. The present invention is described in further detail below with reference to specific examples and with reference to the data. It is to be understood that these examples are for illustrative purposes only and are not to be construed as limiting the scope of the invention, which is defined by the appended claims.
Example 1 construction of a Chicken essence cryopreservation System
The preparation of chicken essence cryopreservation diluent is carried out according to the following components
Preparation of pine needle antifreeze polypeptide liquid: diluting the pine needle antifreeze polypeptide shown in SEQ ID NO.1 in PBS buffer (0.01M pH7.4), and adjusting the concentration to 1mg/ml;
preparation of solution I: 5-10g of fructose, 8-20g of trehalose, 1-3g of potassium citrate monohydrate, 4-6g of magnesium acetate tetrahydrate, 11-19g of sodium glutamate, 8-13g of Tris (Tris (hydroxymethyl aminomethane), 0.1-0.5g of reduced glutathione and 0.05-0.1g of oxidized glutathione are dissolved in 1000ml of water, fully dissolved, then 1-5ml of pine needle antifreeze polypeptide liquid is added to serve as liquid I, filtered and sterilized by a 0.22-micrometer filter, and subpackaged into 2ml of centrifuge tubes for storage;
preparation of liquid II: taking 660ml of the solution I, adding 80-120ml of DMA to obtain a solution II, filtering and sterilizing by using a 0.22um filter, and subpackaging into 2ml centrifuge tubes for storage;
preparing corresponding chicken essence cryopreservation diluent as shown in Table 1
TABLE 1 preparation of chicken essence cryopreservation diluent
Figure DEST_PATH_IMAGE001
The chicken essence liquid freezing preservation method comprises the following steps: firstly, preparing a freezing buffer solution I and a freezing buffer solution II, and carrying out isothermal dilution on the solution I preheated at 37 ℃ and original essence when in use, wherein the original essence: liquid I =1:2, wrapped with gauze, put into a refrigerator, slowly cooled to 4 ℃ within 1h, then added with liquid II for 1:1 dilution, put into a 0.25ml thin tube, frozen by a freezer, the freezing curve is reduced from 5 ℃ to-35 ℃ at 7 ℃/min, and then reduced to-140 ℃ at the rate of-60 ℃/min. The control 5, 6 (control) method is isothermal dilution of preheated buffer with protopine: buffer =1:2, wrapped with gauze and placed in a refrigerator to slowly cool to 4 ℃ within 1h, then added with buffer to dilute 1:1, placed in a 0.25ml thin tube, frozen by a freezer, the freezing curve is decreased from 5 ℃ to-35 ℃ at 7 ℃/min, and then decreased to-140 ℃ at a rate of-60 ℃/min.
The thawing step of the frozen chicken semen comprises the following steps: the thawing temperature is 50 ℃ for 8s.
The unfrozen chicken essence liquid obtained by the different schemes is subjected to corresponding index detection (the freezing preservation time is 10 days),
and (3) vitality examination: diluting 10ul semen with I solution to 100ul (original semen is diluted to 500 ul), and detecting with semen quality analysis system, with the result shown in figure 1 and figure 2.
Fertilization rate of hatching eggs: 700 breeding hens which are 200 days old, healthy, disease-free and high in laying rate are taken for semen transfusion test. The hens are inseminated once every 3 days, each time the injection is 0.2ml, and hatching eggs are collected from the 7 th day after the second injection and are continuously collected for 7 days. The collected hatching eggs are stored under the condition of constant temperature of 18 ℃ and are hatched within 7 days. The hatching mode is carried out by adopting a common artificial hatching procedure, and the hatching rate of hatching eggs is checked on the 21 st day of hatching.
The results of the experiments are shown in table 2,
TABLE 2 frozen chicken semen for corresponding index detection
Figure 32872DEST_PATH_IMAGE002
As is obvious from the data, the pine needle antifreeze polypeptide provided by the application can greatly improve the cryopreservation effect of the chicken semen cryopreservation diluent, and compared with the related data of fresh chicken semen, the sperm motility of the pine needle antifreeze polypeptide is different from the motility (81%) of the fresh semen, but is above 0.60 (0.63-0.72), and reaches and exceeds the standard (0.3) for artificial insemination. And the hatching rate and the DNA integrity far exceed those of a control group, so that the effect of cryopreservation is realized, and even if the control group is subjected to cryopreservation, frozen semen has certain influence on hatching of hatching eggs (the hatching rate is reduced from about 90% to about 80%), but a higher fertilization rate is still obtained after the control group is used, and the fertilization rate is far higher than that of the control group.
The foregoing description of certain exemplary embodiments has been presented for purposes of illustration. Although the foregoing discussion has given specific embodiments, workers skilled in the art will recognize that changes may be made in form and detail without departing from the broader spirit and scope of the invention. The specification and drawings are, accordingly, to be regarded in an illustrative rather than a restrictive sense. The detailed description is, therefore, not to be taken in a limiting sense, and the scope of the present invention is defined only by the appended claims, along with the full scope of equivalents to which such claims are entitled.
(110) Guizhou province breeding livestock and poultry germplasm determination center
<120> antifreeze protein-containing diluent for improving preservation quality of chicken essence and preparation method thereof
<160>1
<170> SIPOSequenceListing 1.0
<210> 1
<211>26
<212> PRT
<213> pine needle antifreeze polypeptide
Ser-Ala-Arg-Glu-Arg-Thr-Cys-Thr-Ser-Ser-Thr-Asn-Ser-Lys-Ala-Cys-Ala-Lys-Tyr-Thr-Pro-Thr-Asp-Thr-Cys-Thr

Claims (4)

1. The diluent for freezing and storing chicken semen is characterized by comprising a solution I and a solution II, wherein the solution I comprises the following components: pine needle antifreeze polypeptide, fructose, trehalose, potassium citrate monohydrate, magnesium acetate tetrahydrate, sodium glutamate, tris Tris (hydroxymethyl) aminomethane, reduced glutathione, oxidized glutathione, wherein the proportion of each component is that, 1000ml system: 1-5ml of pine needle antifreeze polypeptide, 5-10g of fructose, 8-20g of trehalose, 1-3g of potassium citrate monohydrate, 4-6g of magnesium acetate tetrahydrate, 11-19g of sodium glutamate, 8-13g of Tris trihydroxymethyl aminomethane, 0.1-0.5g of reduced glutathione, 0.05-0.1g of oxidized glutathione and the balance of double-distilled water; the amino acid sequence of the pine needle antifreeze polypeptide is shown in SEQ ID NO.1, the initial concentration of the added pine needle antifreeze polypeptide is 1mg/ml, and the pine needle antifreeze polypeptide is dissolved in 0.01M PBS buffer solution;
the liquid II is: 660ml of the solution I is taken and added with 80 to 120ml of dimethylacetamide DMA.
2. The application of the diluent for cryopreservation of chicken semen as claimed in claim 1, which is used for preparing a kit for cryopreservation of chicken semen.
3. A method for cryopreservation of chicken semen using the chicken semen cryopreservation diluent of claim 1, the method comprising the steps of:
dissolving 5-10g of fructose, 8-20g of trehalose, 1-3g of potassium citrate monohydrate, 4-6g of magnesium acetate tetrahydrate, 11-19g of sodium glutamate, 8-13g of Tris (Tris (hydroxymethyl aminomethane)), 0.1-0.5g of reduced glutathione and 0.05-0.1g of oxidized glutathione in 1000ml of water, fully dissolving, adding 1-5ml of pine needle antifreeze polypeptide to obtain a solution I, wherein the initial concentration of the pine needle antifreeze polypeptide is 1mg/ml, and dissolving in 0.01M PBS buffer;
taking 660ml of the solution I, adding 80-120ml of DMA to obtain a solution II, filtering and sterilizing the I, II solution by using a 0.22um filter, and subpackaging the solution into 2ml of centrifuge tubes for storage;
when in use, the solution I is preheated at 37 ℃, and is subjected to isothermal dilution with original essence after being fully and uniformly mixed, wherein the original essence: liquid I =1:2, wrapped with gauze, put into a refrigerator, slowly cooled to 4 ℃ within 1h, then added with liquid II for 1:1 dilution, put into a 0.25ml thin tube, frozen by a freezer, the freezing curve is reduced from 5 ℃ to-35 ℃ at 7 ℃/min, and then reduced to-140 ℃ at a rate of 60 ℃/min.
4. The method of claim 3, further comprising the step of thawing the frozen chicken semen by: thawing in water bath at 50 deg.C for 8s.
CN202210396857.4A 2022-04-16 2022-04-16 Antifreeze protein-containing diluent for improving preservation quality of chicken essence and preparation method thereof Active CN114794082B (en)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN86101836A (en) * 1986-03-22 1987-01-24 中国农业科学院畜牧研究所 Solution for dilution of cock semen
JP2008259506A (en) * 2007-03-20 2008-10-30 Tokyo Univ Of Agriculture Bovine frozen semen for artificial insemination
CN104094926A (en) * 2014-08-05 2014-10-15 燕海峰 Chicken semen diluent and preparation and utilization methods thereof

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN86101836A (en) * 1986-03-22 1987-01-24 中国农业科学院畜牧研究所 Solution for dilution of cock semen
JP2008259506A (en) * 2007-03-20 2008-10-30 Tokyo Univ Of Agriculture Bovine frozen semen for artificial insemination
CN104094926A (en) * 2014-08-05 2014-10-15 燕海峰 Chicken semen diluent and preparation and utilization methods thereof

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