CN113396895A - Application of aloe polysaccharide in preparation of livestock cryopreservation agent - Google Patents
Application of aloe polysaccharide in preparation of livestock cryopreservation agent Download PDFInfo
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Classifications
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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
- A01N1/0215—Disinfecting agents, e.g. antimicrobials for preserving living parts
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- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
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- General Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
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- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
The invention discloses application of aloe polysaccharide in preparation of a livestock semen cryopreservation agent. The invention discloses a scheme that aloe polysaccharide is used for preparing a livestock semen cryoprotectant, is used for semen transportation after long-term preservation of tubule frozen semen of pigs, sheep and cattle, artificial insemination and ultralow temperature semen freezing and can obviously improve the sperm motility rate, acrosome integrity rate and plasma membrane integrity rate of the pigs, sheep and cattle after freezing and thawing.
Description
Technical Field
The invention belongs to the technical field of ultralow temperature cryopreservation of mammal semen, and particularly relates to application of aloe polysaccharide serving as an anti-freezing protective agent in livestock semen cryopreservation.
Background
The sperm freezing technology is an important innovation of artificial insemination, not only solves the problem of long-term preservation of the semen of male animals and is convenient for preserving genetic resources, but also has convenient application, is not limited by time and space, plays an important role in developing the cooperation of inter-provincial and international animal husbandry, establishing a global excellent species gene bank and the like. The wide application of the sperm freezing technology is beneficial to expanding the utilization rate of excellent breeding male livestock, reducing the feeding amount of male livestock, reducing the economic cost, improving the animal husbandry benefit and playing an important role in the field of artificial insemination in particular. The semen freezing technology has important practical significance for sustainable development of animal husbandry and satisfying supply of animal products in China and in China.
The sperm is frozen and stored, a series of programmed cooling treatment is required, and finally the sperm is placed in liquid nitrogen (-198 ℃), and because the sperm is sensitive to temperature change, the sperm is easy to suffer freezing damage in the freezing and cooling process, which mainly comprises physical damage, chemical damage and oxidative damage. Compared with fresh semen, the sperm after freezing and unfreezing has obviously reduced survival rate, acrosome integrity rate, mitochondrial activity, DNA integrity rate and membrane integrity rate. The quality of the sperms is closely related to pregnancy rate, delivery rate and litter size. Up to now, the artificial insemination technology of frozen semen has been applied to the production of cattle raising, and is most popular in the aspects of breeding and improving the dairy cows. But after freezing and thawing, 40 to 50 percent of sperms still lose activity, and the utilization rate of the improved variety bull is seriously reduced. The pig sperms and the sheep sperms are more sensitive to freezing stimulation, the recovery rate of the thawed sperms is low, abnormal sperms are more abundant, and the conception rate is lower than the normal conception rate, so that the pig sperms and the sheep sperms are still in the test stage of freezing.
Disclosure of Invention
Aiming at the defects or shortcomings of the conventional livestock semen freezing, the invention provides the application of aloe polysaccharide in preparing the livestock semen freezing preservative, wherein the livestock is pigs, cattle or sheep.
Furthermore, the invention also provides a livestock semen cryoprotectant. The livestock semen cryoprotectant comprises frozen base liquid, egg yolk and aloe polysaccharide; the addition amount of aloe polysaccharide in 100mL of the livestock semen cryoprotectant is 2mg-60mg, and the addition amount of egg yolk in 100mL of the livestock semen cryoprotectant is 15mL-25 mL; the formula of the freezing base liquid is as follows: 1.1g of glucose, 1.48g of citric acid, 2.42g of Tris2, 0.06g of penicillin sodium, 0.1g of streptomycin sulfate and 100mL of double distilled water.
Another livestock semen cryoprotectant of the invention comprises frozen base liquid, egg yolk, aloe polysaccharide and glycerol; the addition amount of aloe polysaccharide in 100mL of the livestock semen cryoprotectant is 2mg-60mg, the addition amount of egg yolk in 100mL of the livestock semen cryoprotectant is 15mL-25mL, and the addition amount of glycerol in 100mL of the livestock semen cryoprotectant is 6mL-12 mL; the formula of the freezing base liquid is as follows: 1.1g of glucose, 1.48g of citric acid, 2.42g of Tris2, 0.06g of penicillin sodium, 0.1g of streptomycin sulfate and 100mL of double distilled water.
The invention also provides a preparation method of the livestock semen cryoprotectant. The provided method comprises the following steps:
1) dissolving the glucose, the citric acid, the Tris, the penicillin sodium and the streptomycin sulfate in the formula amount in double distilled water, fixing the volume, adjusting the pH to 6-6.5, and preparing a freezing base solution by the osmotic pressure of 280 plus 290 mosm/L;
2) uniformly mixing the frozen base liquid obtained in the step 1) with egg yolk and aloe polysaccharide according to the formula ratio to prepare a liquid I;
3) adding glycerol with the formula amount into the solution I prepared in the step 2), uniformly mixing, adjusting the pH value to 6.0-7.5, adjusting the osmotic pressure to 280-290mosm/L, and sterilizing to prepare a solution II.
Specifically, the addition amount of aloe polysaccharide in 100mL of livestock semen cryoprotectant or I liquid is 4mg-40mg, 2mg-35mg or 6mg-60 mg.
The invention has the beneficial effects that:
the invention uses aloe polysaccharide as freezing antifreeze for freezing and storing the semen of the livestock for the first time, has simple preparation method, easy material obtaining, reliable effect and easy popularization, is suitable for artificial insemination of the tubule frozen semen of the livestock, long-term storage after ultralow temperature freezing of the semen and insemination after long-distance transportation, can obviously improve the motility rate of the sperm, the integrity rate of the acrosome and the integrity rate of the plasma membrane after freezing and thawing, and keeps the fertilization activity of the sperm.
Detailed Description
Unless otherwise indicated, the terms or methods herein are understood to be implemented in accordance with the knowledge of one of ordinary skill or with established correlations.
Through a large number of repeated tests and dose freezing screening, the aloe polysaccharide is found to be capable of obviously improving the sperm motility rate, the membrane integrity and the like after freezing in the livestock sperm freezing protection, particularly after the sperm of the livestock such as pigs, sheep, cattle and the like are frozen and stored.
Based on the scheme of the invention, a person skilled in the art can optimize related parameters such as the addition amount of substances, the pH value, the osmotic pressure and the like in the livestock semen cryoprotectant in the specific scheme by adopting a conventional experimental method, and the optimized methods can achieve the purpose of the invention and belong to the protection scope of the invention.
The present invention will be described in detail with reference to specific examples.
In the following examples, astragalus polysaccharide is simultaneously selected as a control, is the main active component with the most research and efficacy in astragalus and has various effects of resisting aging, oxidation, viruses, apoptosis and stress, and the like. The results suggest that aloe polysaccharide has a significant protective effect in the preservation of frozen semen of domestic animals, especially in the protection of semen of boars, compared with astragalus polysaccharide.
Aloe polysaccharide and Astragalus polysaccharide used in the following examples and comparative examples were purchased from Shanghai-derived leaf Biotech, Inc. and stored at 2-8 deg.C.
Example 1:
the preparation method of the frozen pig semen preservative comprises the following steps:
step one, preparing a freezing base liquid: accurately measuring 1.1g of glucose, 1.48g of citric acid, 2.42g of Tris2, 0.06g of penicillin sodium and 0.1g of streptomycin sulfate, dissolving in 100ml of double distilled water, adjusting the pH value to 6.21, and preparing into a frozen base solution under the osmotic pressure of 286 mosm/L;
step two, accurately measuring 80ml of frozen base solution, adding 20ml of fresh egg yolk and 40mg of aloe polysaccharide sugar, and uniformly mixing to obtain solution I;
and step three, adding 6ml of glycerol into 94ml of the solution I, uniformly mixing, adjusting the pH value to 6.0-7.5, adjusting the osmotic pressure to 286mosm/L, filtering, sterilizing and cooling to room temperature to obtain a solution II. The test specimens were kept in a 4 ℃ freezer for further testing.
The inventors of the present example conducted the following tests on the effect of using the diluent for cryopreservation of livestock semen.
(I) porcine semen collection
Collecting semen by hand-held method, putting sterilized gloves on operator, sterilizing penis, collecting middle thick semen, filtering with 4 layers of sterilized gauze to remove jelly, and performing routine quality inspection of semen at 35-37 deg.C with microscope; semen with no foreign taste, milky white color, normal sperm morphology and a survival rate of more than 0.7 is selected for the test.
(II) semen treatment and freezing
Adding fresh boar semen into isothermal BTS diluent (diluted by 1: 1 at 30 ℃), wrapping with 8-10 gauze, and balancing in a thermostat at 17 ℃ for 30 min;
centrifuging the semen after the balance treatment at 17 ℃ (1500 Xg for 5min), discarding the supernatant, adding the solution I with isothermal volume twice that of the embodiment, wrapping the solution I with gauze, and balancing the solution in a refrigerator at 4 ℃ for 1-2 h;
then adding the semen subjected to balance treatment into the equal volume of the solution II at 4 ℃, wrapping the solution with gauze, and then placing the wrapped solution in a refrigerator at 4 ℃ for balancing for 2-3 hours;
adjusting sperm density to 1.0 × 109And (2) sucking the semen into a 0.25ml thin tube by using a special syringe, quickly sealing the tube, slowly reducing the temperature from 4 ℃ to-5 ℃ in a program freezing instrument at the speed of 1 ℃/min, quickly moving to the position 3cm above liquid nitrogen, fumigating for 10min, and putting the thin tube into the liquid nitrogen for preservation.
(III) thawing of tubule frozen semen and quality evaluation of semen
1. Motility rate of sperm
Taking out frozen semen of the thin tube, rapidly placing the frozen semen into a water bath at 37 ℃ for thawing for 45s, collecting the frozen semen in a small test tube, diluting the frozen semen with a normal-temperature diluent, incubating for 5min, taking 10 mu L of semen on a glass slide, covering a cover glass, and evaluating the sperm motility rate (percentage of linearly moving sperm) under a 400X inverted microscope; at least 200 sperm cells were examined each time and repeated 5 times.
2. Percentage of sperm acrosome integrity
After staining with FITC-PNA staining solution, fluorescent development was performedObserving the shape of the sperm acrosome by a micro-mirror; thawing frozen semen in a thin tube, adding semen onto 2ml of 3% PVP liquid surface, centrifuging and washing for 2 times at 800 Xg for 3min, and removing supernatant; resuspending in PBS (37 ℃) to adjust the density to 1-2 xl 06Sperm/ml; sucking 30 μ l of sperm suspension, smearing, air drying, and fixing with pure methanol for 10 min; then adding 30 mul FITC-PNA dye solution, and incubating for 30min in a dark and humid environment at 37 ℃; PBS rinse, air dry, drop small amount of glycerol: PBS (9: 1), coverslipping, mounting with colorless nail polish, and viewing as soon as possible under 400 Xfluorescence microscope; at least 200 sperm cells were examined each time and repeated 5 times.
3. Sperm plasma membrane integrity rate
Hypotonic swelling assay (HOST) was performed using fructose-sodium citrate hypotonic solution: diluting the thawed semen with conventional diluent to adjust sperm density to 1 × L06Incubating at 37 deg.C for 30min, dropping 20 μ l semen into suspension, observing under 400 × microscope, calculating percentage of curved tail sperm, checking at least 200 sperm each time, and repeating for 5 times.
(IV) sperm quality assessment results
The livestock semen cryopreservation agent of the embodiment is used for preserving the pig sperms, and the semen freezing-unfreezing method is adopted, so that the freezing preservation effect is shown in table 1.
Comparative example 1:
the comparative example differs from the example in that aloe polysaccharides are replaced by astragalus polysaccharides. The livestock cryopreservation agent of this comparative example exhibited cryopreservation effects on pig sperm as shown in table 1.
TABLE 1 quality change of frozen boar semen
Example 2:
the preparation method of the sheep semen cryopreservation agent comprises the following steps:
step one, preparing a freezing base solution by the same method as the example 1;
step two, accurately measuring 85mL of the frozen base solution, adding 15mL of fresh egg yolk and 30mg of aloe polysaccharide, and uniformly mixing to prepare solution I of the embodiment;
and step three, adding 6mL of glycerol into 94mL of the solution I to prepare 100mL of mixed solution, adjusting the pH value and the osmotic pressure, filtering, sterilizing, and cooling to room temperature to obtain the solution II of the embodiment. The test pieces were placed in a 4 ℃ freezer for later use.
(I) sheep sperm collection
Sperm were collected by the pseudo-vaginal method and examined for routine quality using a microscope at 37 ℃. Semen with no foreign odor, milky color, normal sperm morphology, a survival rate of above 0.7 and a density of dense is selected for the test.
(II) semen treatment and freezing
Adding fresh sheep semen into the frozen base solution (diluted at 30 ℃, 1: 3), wrapping with 8-10 gauze, and balancing in a thermostat at 17 ℃ for 30 min;
centrifuging the semen after the balance treatment at 17 ℃ (1500 Xg for 5min), discarding the supernatant, adding the solution I with isothermal volume twice that of the embodiment, wrapping the solution I with gauze, and balancing the solution in a refrigerator at 4 ℃ for 1-2 h;
then adding the semen subjected to balance treatment into the equal volume of the solution II at 4 ℃, wrapping the solution with gauze, and then placing the wrapped solution in a refrigerator at 4 ℃ for balancing for 2-3 hours;
adjusting sperm density to 1.0 × 109Sucking semen into 0.25ml tube with special syringe, rapidly sealing tube, slowly cooling from 4 deg.C to-5 deg.C at 1 deg.C/min, rapidly transferring to 2-3cm above liquid nitrogen, fumigating for 5-10min, and storing in liquid nitrogen.
(III) thawing of tubule frozen semen and quality evaluation of semen
1. Motility rate of sperm
Taking out frozen semen of the thin tube, rapidly placing the frozen semen into a water bath at 37 ℃ for thawing for 45s, collecting the frozen semen in a small test tube, diluting the frozen semen with a normal-temperature diluent, incubating for 5min, taking 10 mu L of semen on a glass slide, covering a cover glass, and evaluating the sperm motility rate (percentage of linearly moving sperm) under a 400X inverted microscope; at least 200 sperm cells were examined each time and repeated 5 times.
2. Percentage of sperm acrosome integrity
After staining with FITC-PNA staining solution, sperm acrosome morphology was observed with a fluorescence microscope. Thawing frozen semen in a thin tube, adding semen onto 2ml of 3% PVP liquid surface, centrifuging and washing for 2 times at 800 Xg for 3min, and removing supernatant; resuspending in PBS (37 ℃) to adjust the density to 1-2X 106Sperm/ml; sucking 30 μ l of sperm suspension, smearing, air drying, and fixing with pure methanol for 10 min; then adding 30 mul FITC-PNA dye solution, and incubating for 30min in a dark and humid environment at 37 ℃; PBS rinse, air dry, drop small amount of glycerol: PBS (9: 1), coverslipping, mounting with colorless nail polish, and viewing as soon as possible under 400 Xfluorescence microscope; at least 200 sperm cells were examined each time and repeated 5 times.
3. Sperm plasma membrane integrity rate
Hypotonic swelling assay (HOST) was performed using fructose-sodium citrate hypotonic solution: diluting the thawed semen with conventional diluent to adjust sperm density to 1 × L06Incubating at 37 deg.C for 30min, dropping 20 μ l semen into suspension, observing under 400 × microscope, calculating percentage of curved tail sperm, checking at least 200 sperm each time, and repeating for 5 times.
(IV) sperm quality assessment results
The livestock semen cryopreservation agent of the embodiment is used for preserving sheep semen, and a semen freezing-unfreezing method is adopted, so that the freezing preservation effect is shown in table 2.
Comparative example 2:
the comparative example is different from example 2 in that aloe polysaccharide was replaced with astragalus polysaccharide, and the livestock cryopreservative of the comparative example has the cryopreservation effect on sheep semen as shown in table 1.
TABLE 2
Example 3:
the preparation method of the bovine semen cryopreservation agent comprises the following steps:
step one, preparing a freezing base solution by the same method as the example 1;
step two, accurately measuring 80ml of frozen base liquid, adding 20ml of fresh egg yolk and 50mg of aloe polysaccharide to obtain the liquid I of the embodiment;
and step three, adding 6mL of glycerol into the 94mlI solution, uniformly mixing, adjusting the pH value and the osmotic pressure, filtering, sterilizing and cooling to room temperature to obtain the solution II of the embodiment. The test pieces were placed in a 4 ℃ freezer for later use.
(I) bovine sperm collection
Sperm were collected by the pseudo-vaginal method and examined for routine quality using a microscope at 37 ℃. Semen with no foreign taste, milky white color, normal sperm morphology and a survival rate of more than 0.7 is selected for the test.
(II) semen treatment and freezing
Adding fresh bovine semen into the frozen base liquid (heated in advance to 30 deg.C), and adjusting semen density to 1.5 × 106Each/ml is wrapped by gauze and then balanced in a refrigerator at 4 ℃ for 3 hours; wrapping with 8-10 gauze, and balancing in a thermostat at 17 ℃ for 30 min;
centrifuging the semen after the balance treatment at 17 ℃ (1500 Xg for 5min), discarding the supernatant, adding the solution I with isothermal volume twice that of the embodiment, wrapping the solution I with gauze, and balancing the solution in a refrigerator at 4 ℃ for 1-2 h;
then adding the semen subjected to balance treatment into the equal volume of the solution II at 4 ℃, wrapping the solution with gauze, and then placing the wrapped solution in a refrigerator at 4 ℃ for balancing for 2-3 hours;
sucking the semen after balanced treatment into a 0.25ml tube by a special syringe, quickly sealing the tube, slowly cooling from 4 ℃ to-5 ℃ in a program freezing instrument at the speed of 1 ℃/min, quickly moving to 2-3cm above liquid nitrogen, fumigating for 5-10min, and putting the tube into the liquid nitrogen for storage.
(III) thawing of tubule frozen semen and quality evaluation of semen
1. Motility rate of sperm
Taking out frozen semen of the thin tube, rapidly placing the frozen semen into a water bath at 37 ℃ for thawing for 45s, collecting the frozen semen in a small test tube, diluting the frozen semen with a normal-temperature diluent in an equivalent manner, incubating for 5min, taking 10 mu L of semen on a glass slide, covering the glass slide, and evaluating the sperm motility rate (percentage of linearly moving sperm) under a 400X inverted microscope; at least 200 sperm cells were examined each time and repeated 5 times.
2. Percentage of sperm acrosome integrity
After dyeing by using FITC-PNA dye solution, observing the shape of a sperm acrosome by using a fluorescence microscope, unfreezing a tubule, adding the semen onto a 2ml 3% PVP liquid surface, carrying out centrifugal washing for 2 times at 800 Xg for 3min, and removing the supernatant; resuspending in PBS (37 ℃) to adjust the density to 1-2 xl 06Sperm/ml; sucking 30 μ l of sperm suspension, smearing, air drying, and fixing with pure methanol for 10 min; then adding 30 mul FITC-PNA dye solution, and incubating for 30min in a dark and humid environment at 37 ℃; PBS rinse, air dry, drop small amount of glycerol: PBS (9: 1), coverslipping, mounting with colorless nail polish, and viewing as soon as possible under 400 Xfluorescence microscope; at least 200 sperm cells were examined each time and repeated 5 times.
3. Sperm plasma membrane integrity rate
Hypotonic swelling assay (HOST) was performed using fructose-sodium citrate hypotonic solution: diluting the thawed semen with conventional diluent, adjusting sperm density to 1 × 106/ml, incubating at 37 deg.C for 30min, dripping 20 μ l semen into suspension on a hemocytometer, observing under 400 × microscope, calculating percentage of bent-tail sperm, checking at least 200 sperm each time, and repeating for 5 times.
(IV) sperm quality assessment results
The livestock semen cryopreservation agent of the embodiment is used for preserving bovine sperms, and the semen freezing-unfreezing method is adopted, so that the freezing preservation effect is shown in Table 3.
Comparative example 3:
this comparative example differs from example 3 in that aloe polysaccharides are replaced by astragalus polysaccharides. The livestock cryopreservation agent of this comparative example exhibited cryopreservation effects on bovine sperm as shown in Table 3.
TABLE 3
It will be understood that modifications and variations can be made by persons skilled in the art in light of the above teachings and all such modifications and variations are intended to be included within the scope of the invention as defined in the appended claims.
Claims (9)
1. The application of aloe polysaccharide in preparing the freeze preservative for the semen of domestic animals such as pig, cattle or sheep.
2. The livestock semen cryoprotectant is characterized by comprising frozen base liquid, egg yolk and aloe polysaccharide; the addition amount of aloe polysaccharide in 100mL of the livestock semen cryoprotectant is 2mg-60mg, and the addition amount of egg yolk in 100mL of the livestock semen cryoprotectant is 15mL-25 mL; the formula of the freezing base liquid is as follows: 1.1g of glucose, 1.48g of citric acid, 2.42g of Tris2, 0.06g of penicillin sodium, 0.1g of streptomycin sulfate and 100mL of double distilled water.
3. The livestock semen cryoprotectant is characterized by comprising frozen base liquid, egg yolk, aloe polysaccharide and glycerol; the addition amount of aloe polysaccharide in 100mL of the livestock semen cryoprotectant is 2mg-60mg, the addition amount of egg yolk in 100mL of the livestock semen cryoprotectant is 15mL-25mL, and the addition amount of glycerol in 100mL of the livestock semen cryoprotectant is 6mL-12 mL; the formula of the freezing base liquid is as follows: 1.1g of glucose, 1.48g of citric acid, 2.42g of Tris2, 0.06g of penicillin sodium, 0.1g of streptomycin sulfate and 100mL of double distilled water.
4. A livestock semen cryoprotectant is characterized by comprising a freezing base solution, a solution I and a solution II which are respectively and independently packaged;
the formula of the freezing base liquid is as follows: 1.1g of glucose, 1.48g of citric acid, 2.42g of Tris2, 0.06g of penicillin sodium, 0.1g of streptomycin sulfate and 100mL of double distilled water;
the solution I is prepared from frozen base solution, egg yolk and aloe polysaccharide, wherein the addition amount of the aloe polysaccharide in 100mL of the solution I is 2mg-60mg, and the addition amount of the egg yolk in 100mL of the solution I is 15mL-25 mL;
the liquid II is prepared from the liquid I and glycerol; the addition amount of glycerol in 100mL of the II solution is 6mL-12 mL.
5. A livestock semen cryoprotectant is characterized by comprising a liquid I and a liquid II which are respectively and independently packaged;
the solution I is prepared from frozen base solution, egg yolk and aloe polysaccharide, wherein the addition amount of the aloe polysaccharide in 100mL of the solution I is 2mg-60mg, and the addition amount of the egg yolk in 100mL of the solution I is 15mL-25 mL; the formula of the freezing base liquid is as follows: 1.1g of glucose, 1.48g of citric acid, 2.42g of Tris2, 0.06g of penicillin sodium, 0.1g of streptomycin sulfate and 100mL of double distilled water;
the liquid II is prepared from the liquid I and glycerol; the addition amount of glycerol in 100mL of the II solution is 6mL-12 mL.
6. A livestock semen cryoprotectant as claimed in any one of claims 2 to 5, wherein the amount of aloe polysaccharide added to 100mL of the livestock semen cryoprotectant or I solution is 4mg to 40 mg.
7. A livestock semen cryoprotectant as claimed in any one of claims 2 to 5, wherein the amount of aloe polysaccharide added to 100mL of the livestock semen cryoprotectant or I solution is 2mg to 35 mg.
8. A livestock semen cryoprotectant as claimed in any one of claims 2 to 5, wherein the amount of aloe polysaccharide added to 100mL of the livestock semen cryoprotectant or I solution is 6mg to 60 mg.
9. A method of producing a cryoprotectant for livestock semen as claimed in any one of claims 2 to 5, the method comprising:
1) dissolving the glucose, the citric acid, the Tris, the penicillin sodium and the streptomycin sulfate in the formula amount in double distilled water, fixing the volume, adjusting the pH to 6-6.5, and preparing a freezing base solution by the osmotic pressure of 280 plus 290 mosm/L;
2) uniformly mixing the frozen base liquid obtained in the step 1) with egg yolk and aloe polysaccharide according to the formula ratio to prepare a liquid I;
3) adding glycerol with the formula amount into the solution I prepared in the step 2), uniformly mixing, adjusting the pH value to 6.0-7.5, adjusting the osmotic pressure to 280-290mosm/L, and sterilizing to prepare a solution II.
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