CN114651815A - Method for preserving frozen boar semen - Google Patents
Method for preserving frozen boar semen Download PDFInfo
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- CN114651815A CN114651815A CN202210381511.7A CN202210381511A CN114651815A CN 114651815 A CN114651815 A CN 114651815A CN 202210381511 A CN202210381511 A CN 202210381511A CN 114651815 A CN114651815 A CN 114651815A
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- China
- Prior art keywords
- semen
- water
- diluent
- polyglutamic acid
- lactose
- Prior art date
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- Granted
Links
- 210000000582 semen Anatomy 0.000 title claims abstract description 60
- 238000000034 method Methods 0.000 title claims abstract description 55
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 75
- 239000003085 diluting agent Substances 0.000 claims abstract description 55
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims abstract description 45
- 108010020346 Polyglutamic Acid Proteins 0.000 claims abstract description 33
- 229920002643 polyglutamic acid Polymers 0.000 claims abstract description 33
- 230000008569 process Effects 0.000 claims abstract description 30
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 claims abstract description 29
- 239000008101 lactose Substances 0.000 claims abstract description 29
- 102000005936 beta-Galactosidase Human genes 0.000 claims abstract description 21
- 108010005774 beta-Galactosidase Proteins 0.000 claims abstract description 21
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 21
- 238000002156 mixing Methods 0.000 claims abstract description 19
- 230000008014 freezing Effects 0.000 claims abstract description 17
- 238000004321 preservation Methods 0.000 claims abstract description 14
- 238000010790 dilution Methods 0.000 claims abstract description 5
- 239000012895 dilution Substances 0.000 claims abstract description 5
- 210000002969 egg yolk Anatomy 0.000 claims description 32
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 26
- 239000011734 sodium Substances 0.000 claims description 24
- DRBBFCLWYRJSJZ-UHFFFAOYSA-N N-phosphocreatine Chemical compound OC(=O)CN(C)C(=N)NP(O)(O)=O DRBBFCLWYRJSJZ-UHFFFAOYSA-N 0.000 claims description 22
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 15
- 239000007983 Tris buffer Substances 0.000 claims description 15
- 235000018417 cysteine Nutrition 0.000 claims description 15
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 claims description 15
- 239000008103 glucose Substances 0.000 claims description 15
- 239000001509 sodium citrate Substances 0.000 claims description 15
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 claims description 15
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims description 15
- 239000011780 sodium chloride Substances 0.000 claims description 13
- 102000002322 Egg Proteins Human genes 0.000 claims description 11
- 108010000912 Egg Proteins Proteins 0.000 claims description 11
- 235000013345 egg yolk Nutrition 0.000 claims description 11
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 10
- 235000001014 amino acid Nutrition 0.000 claims description 10
- 150000001413 amino acids Chemical class 0.000 claims description 10
- 229910052708 sodium Inorganic materials 0.000 claims description 10
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 claims description 8
- 239000007788 liquid Substances 0.000 claims description 7
- 238000001816 cooling Methods 0.000 claims description 5
- 229910052757 nitrogen Inorganic materials 0.000 claims description 5
- 238000002360 preparation method Methods 0.000 claims description 4
- 239000006228 supernatant Substances 0.000 claims description 3
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 claims description 2
- 238000002203 pretreatment Methods 0.000 claims description 2
- ZGTMUACCHSMWAC-UHFFFAOYSA-L EDTA disodium salt (anhydrous) Chemical compound [Na+].[Na+].OC(=O)CN(CC([O-])=O)CCN(CC(O)=O)CC([O-])=O ZGTMUACCHSMWAC-UHFFFAOYSA-L 0.000 claims 1
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 claims 1
- 238000007710 freezing Methods 0.000 abstract description 12
- 210000000170 cell membrane Anatomy 0.000 abstract description 9
- 239000013078 crystal Substances 0.000 abstract description 8
- 241000282887 Suidae Species 0.000 abstract description 6
- 230000000694 effects Effects 0.000 abstract description 5
- 230000009027 insemination Effects 0.000 abstract description 4
- 239000012530 fluid Substances 0.000 abstract description 2
- 244000144972 livestock Species 0.000 abstract description 2
- UNFWWIHTNXNPBV-WXKVUWSESA-N spectinomycin Chemical compound O([C@@H]1[C@@H](NC)[C@@H](O)[C@H]([C@@H]([C@H]1O1)O)NC)[C@]2(O)[C@H]1O[C@H](C)CC2=O UNFWWIHTNXNPBV-WXKVUWSESA-N 0.000 description 14
- 229960000268 spectinomycin Drugs 0.000 description 14
- 238000006243 chemical reaction Methods 0.000 description 11
- 230000000052 comparative effect Effects 0.000 description 10
- 239000000047 product Substances 0.000 description 8
- 230000019100 sperm motility Effects 0.000 description 7
- 210000004027 cell Anatomy 0.000 description 6
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 5
- 230000018044 dehydration Effects 0.000 description 4
- 238000006297 dehydration reaction Methods 0.000 description 4
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 3
- 238000001035 drying Methods 0.000 description 3
- 230000004720 fertilization Effects 0.000 description 3
- 230000009467 reduction Effects 0.000 description 3
- 239000004382 Amylase Substances 0.000 description 2
- 102000013142 Amylases Human genes 0.000 description 2
- 108010065511 Amylases Proteins 0.000 description 2
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 2
- 229920002472 Starch Polymers 0.000 description 2
- 235000019418 amylase Nutrition 0.000 description 2
- 238000009395 breeding Methods 0.000 description 2
- 230000001488 breeding effect Effects 0.000 description 2
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 2
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 235000021255 galacto-oligosaccharides Nutrition 0.000 description 2
- 150000003271 galactooligosaccharides Chemical class 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- 239000001257 hydrogen Substances 0.000 description 2
- 229910052739 hydrogen Inorganic materials 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004899 motility Effects 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 239000008107 starch Substances 0.000 description 2
- 235000019698 starch Nutrition 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 238000010257 thawing Methods 0.000 description 2
- 241001138401 Kluyveromyces lactis Species 0.000 description 1
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000030833 cell death Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 235000012000 cholesterol Nutrition 0.000 description 1
- 239000002577 cryoprotective agent Substances 0.000 description 1
- 238000002425 crystallisation Methods 0.000 description 1
- 230000008025 crystallization Effects 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 230000002427 irreversible effect Effects 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 230000013011 mating Effects 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 230000035515 penetration Effects 0.000 description 1
- 239000000546 pharmaceutical excipient Substances 0.000 description 1
- 229940124531 pharmaceutical excipient Drugs 0.000 description 1
- 239000008055 phosphate buffer solution Substances 0.000 description 1
- 150000003904 phospholipids Chemical class 0.000 description 1
- 230000035935 pregnancy Effects 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000001632 sodium acetate Substances 0.000 description 1
- 235000017281 sodium acetate Nutrition 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 239000012192 staining solution Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 230000035899 viability Effects 0.000 description 1
- 230000000007 visual effect Effects 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Classifications
-
- 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
-
- 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
-
- 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
-
- 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
- A01N1/0278—Physical preservation processes
- A01N1/0284—Temperature processes, i.e. using a designated change in temperature over time
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Dentistry (AREA)
- General Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Environmental Sciences (AREA)
- Biophysics (AREA)
- Physiology (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
The invention relates to the technical field of livestock artificial insemination, and relates to a method for preserving frozen boar semen. The preservation method comprises the following steps: mixing the pig semen in a pretreatment agent for pretreatment to obtain pretreated semen; mixing the pretreated semen with a diluent at 0-3 ℃ for dilution to obtain diluted semen; carrying out program freezing on the diluted semen to obtain frozen semen; wherein the diluent comprises, based on 100g of water: 5-12g of glycerol, 3-8g of PVA, 5-12g of polyglutamic acid, 0.5-8g of lactose, 0.02-0.08g of beta-galactosidase and 100g of water. According to the invention, the specific pretreatment agent is combined with the specific refrigerating fluid, so that the living activity of the spermatids is not influenced, and the content of free water in the spermatids and outside the spermatids is reduced, thereby preventing the part of free water from crystallizing in the freezing process, and simultaneously preventing the crystals from further coarsening and growing to further puncture a plasma membrane, thereby protecting the sperms of the pigs from being damaged.
Description
Technical Field
The invention relates to the technical field of livestock artificial insemination, and relates to a method for preserving frozen boar semen.
Background
At present, the technology of preserving semen in a liquid state at normal temperature for pigs is widely popularized and applied to pig raising production practice. The freezing preservation of the boar semen solves the problem of long-term preservation of the semen and is beneficial to improving the utilization rate of excellent boars; and the service life of the genetic material of the breeding pigs is greatly prolonged, so that the excellent breeding pigs have important meanings in the aspects of short-term descendant determination, semen supply reservation and recovery, pedigree renewal, introduction, production cost reduction and the like.
Because the pig sperms are sensitive to the processes of temperature reduction, freezing and unfreezing, the types, concentrations, dilution ratios, freezing rates, unfreezing methods and the like of diluent components and cryoprotectants influence the sperm motility in the process of freezing the pig sperms, and finally the sperm motility and the fertilization capability are reduced, so that the artificial insemination effect of the frozen semen is far inferior to that of fresh sperm insemination and natural mating.
Because the plasma membrane structure of the sperms of the pig is changed in the freezing process, the recovery rate of the sperms after thawing is low, and further the pregnancy rate of the sow is reduced and the litter size is small.
Disclosure of Invention
The invention aims to overcome the problem of poor sperm motility and fertilization capability of frozen pigs in the prior art and provide a method for preserving frozen boar semen.
In order to achieve the aim, the invention provides a method for preserving frozen pig semen, which comprises the following steps:
mixing the pig semen in a pretreatment agent for pretreatment to obtain pretreated semen;
mixing the pre-treated semen with a diluent for dilution to obtain diluted semen;
carrying out program freezing on the diluted semen to obtain frozen semen;
wherein the diluent comprises, expressed as 100g of water: 5-12g of glycerol, 3-8g of PVA, 5-12g of polyglutamic acid, 0.5-8g of lactose, 0.02-0.08g of beta-galactosidase and 100g of water.
The invention discovers that part of the reason for changing the plasma membrane structure of the sperms is that trace moisture attached outside the sperms can crystallize and generate ice crystals under low temperature; ice crystals puncture the sperm plasma membrane, which in turn causes a change in the structure of the plasma membrane, loss of phospholipids and cholesterol within the sperm cell, and ultimately a decrease in sperm motility and fertilization. The inventor of the invention researches for a long time to find that the product molecules generated by the reaction of polyglutamic acid, lactose and beta-galactosidase contain a large amount of hydroxyl (derived from lactose), and the hydroxyl can capture water molecules on the surface of sperms and form hydrogen bonds, so that the water molecules can not be frozen; on the other hand, product molecules can be combined on the surface of the ice crystal nucleus to limit the further growth of the crystal nucleus and reduce the penetration of the ice crystal into a plasma membrane; in addition, the modified product can fix water molecules on the surface of sperms, and water molecules on the surface of sperms cannot be lost, so that the phenomena of cell dehydration and other chemical damages caused by rapid reduction of extracellular water in the freezing process can be reduced.
In some preferred embodiments of the present invention, to further reduce damage to the plasma membrane of the sperm, preferably, the diluent comprises, based on 100g of water: 5-12g of glycerol, 3-8g of PVA, 6-10g of polyglutamic acid, 0.6-3g of lactose, 0.02-0.08g of beta-galactosidase and 100g of water.
According to the invention, preferably, the diluent further comprises, based on 100g of water: 22-30g of glucose, 4-8g of sodium citrate, 2-3.2g of sodium N-2-hydroxyethylpiperazine-N' -2-ethanesulfonate, 4-7g of Tris and 1.5-4g of EDTA-Na20.1-0.5g of cysteine and 20-50g of egg yolk diluent.
In some preferred embodiments of the invention, the egg yolk diluent consists of water, egg yolk and spectinomycin in a ratio of 1-2:1: 0.3-0.8. Under the above-described preferable conditions, the activity of the thawed sperm can be further improved.
According to the present invention, preferably, the method further comprises the preparation of a diluent, wherein the method of preparing the diluent comprises: mixing polyglutamic acid, lactose and beta-galactosidase in water, reacting at 20-40 deg.C for 5-40h with pH of 3-5, and adding glucose, sodium citrate, N-2-hydroxyethyl piperazine-N' -2-ethanesulfonic acid sodium salt, Tris, EDTA-Na2And uniformly mixing cysteine, yolk diluent and the rest water to obtain the diluent.
According to the invention, lactose can be hydrolyzed under the action of beta-galactosidase to generate galactooligosaccharide, and the galactooligosaccharide further reacts with polyglutamic acid to generate a product with a long-chain structure containing a large number of hydroxyl and carboxyl; hydroxyl in the product molecule can form hydrogen bonds with water molecules on the surface of the sperms, and carboxyl can perform intermolecular dehydration with the hydroxyl in the PVA, so that the PVA is coated on the surface of the product, the water balance of spermatids is maintained, and the damage of spermatid membranes in the freezing process is reduced.
According to the invention, the weight ratio of the lactose to the polyglutamic acid is 1:1-10 under the preferable conditions.
According to the present invention, the molecular weight of the polyglutamic acid is preferably 7 to 10 ten thousand.
According to the present invention, the molecular weight of the PVA is 20000-220000 under the preferable conditions.
In some preferred embodiments of the invention, the pre-treatment comprises: and (3) placing the boar semen in the pretreating agent for standing for 0.2-2h at the temperature of 18-22 ℃, then centrifuging, and removing a supernatant to obtain the pretreated semen.
According to the invention, under preferred conditions, the pretreatment agent comprises, based on 100g of water: 2-5g of sodium chloride, 3-8mM of amino acid, 0.1-0.5g of creatine phosphate and 100g of water.
In the present invention, by pretreating pig sperm in an aqueous solution containing sodium chloride and creatine phosphate, sperm can be rendered active in a high concentration permeation solution, cells thereof can be dehydrated to a certain extent, and the water content in the cells can be reduced.
In the invention, the concentration of the sodium chloride is particularly important, when the content of the sodium chloride is too high, irreversible cell dehydration death of spermatids can be caused, and when the concentration is too low, the effect of effective dehydration cannot be achieved.
In the invention, under the preferable conditions, the volume ratio of the pig semen to the pretreating agent is 1: 3-8.
According to the present invention, preferably, the method of program freezing comprises: cooling the diluted semen to 0-5 ℃ at the speed of 1-2 ℃/min, preserving the heat for 10-30min, and then putting the diluted semen into liquid nitrogen to cool to-196 ℃.
Through the technical scheme, the special pretreatment agent is combined with the special refrigerating fluid, so that the living activity of the spermatids is not influenced, the content of free water in the spermatids and outside the spermatids is reduced, the crystallization of the part of the free water in the freezing process is avoided, the crystals are prevented from further coarsening and growing, the plasma membrane is further punctured, and the pig sperms are protected from being damaged.
And the special freezing method is combined, so that the cells rapidly cross a cell death danger temperature area (-15 to-60 ℃) in the freezing process, the formation of ice crystals is further reduced, and the survival rate of the spermatids is improved.
Detailed Description
The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value, and such ranges or values should be understood to encompass values close to those ranges or values. For ranges of values, between the endpoints of each of the ranges and the individual points, and between the individual points may be combined with each other to give one or more new ranges of values, and these ranges of values should be considered as specifically disclosed herein.
The present invention will be described in detail below by way of examples.
In the following examples, polyglutamic acid is a product sold by science and technology limited of coanda in Xian and has a molecular weight of 70 ten thousand, and PVA is a product sold by pure pharmaceutical excipients limited of Xian Tian and has a molecular weight of 2 to 22 ten thousand; the beta-galactosidase is Kluyveromyces lactis.
Example 1
(1) Preparation of the reagent:
the pretreatment agent comprises the following components: 3g of sodium chloride, 5mM of amino acids, 0.2g of creatine phosphate and 100g of water;
preparation of the diluent: mixing polyglutamic acid 8g, lactose 2g (weight ratio of lactose to polyglutamic acid is 1:4) and beta-galactosidase 0.04g in water 30g, reacting at 30 deg.C for 24h, pH 3.5, adding PVA 5g, continuing reaction for 20min, adding glycerol 8g, glucose 25g, sodium citrate 4.5g, sodium N-2-hydroxyethyl piperazine-N' -2-ethanesulfonate 2.8g, Tris 5g, and EDTA-Na 2.5g20.2g of cysteine, 30g of egg yolk diluent (consisting of water, egg yolk and spectinomycin according to a ratio of 1:1: 0.5) and 70g of water are uniformly mixed to obtain the diluent.
(2) The preservation method of the frozen pig semen comprises the following steps:
standing the boar semen in a pretreating agent for 10 hours at the temperature of 20 ℃, then centrifuging, and removing supernatant to obtain pretreated semen; the volume ratio of the pre-pretreatment agent of the pig semen is 1: 5;
mixing the pre-treated semen with a diluent according to the volume ratio of 1:1 for dilution to obtain diluted semen;
cooling the diluted semen to 0-5 deg.C at a rate of 1-2 deg.C/min, maintaining the temperature for 10-30min, and cooling to-196 deg.C in liquid nitrogen to obtain frozen semen.
Example 2
The process of example 1 was followed except that: the pretreatment agent comprises the following components: 2g of sodium chloride, 8mM of amino acids, 0.5g of creatine phosphate and 100g of water.
Example 3
The process of example 1 was followed except that: the pretreatment agent comprises the following components: 5g of sodium chloride, 3mM of amino acids, 0.1g of creatine phosphate and 100g of water.
Example 4
The process of example 1 was followed except that: the pretreatment agent comprises the following components: 3g of sodium chloride, 5mM of amino acids, 0.2g of creatine phosphate and 100g of water.
Comparative example 1
The process of example 1 was followed except that: the pretreatment agent comprises the following components: 0.5g of sodium chloride, 5mM of amino acids, 0.2g of creatine phosphate and 100g of water.
Comparative example 2
The process of example 1 was followed except that: the pretreatment agent comprises the following components: 15g of sodium chloride, 5mM of amino acids, 0.2g of creatine phosphate and 100g of water.
Comparative example 3
The process of example 1 was followed except that: the pretreatment agent comprises the following components: 2g of sodium chloride, 5mM of amino acids, 0.01g of creatine phosphate and 100g of water.
Comparative example 4
The process of example 1 was followed except that: the pretreatment agent comprises the following components: 2g of sodium chloride, 5mM of amino acids, 5g of creatine phosphate and 100g of water.
Test example
The method comprises the steps of freezing fresh collected boar semen A for 10 days by adopting the methods of examples 1-4 and comparative examples 1-4, then unfreezing the frozen boar semen in warm water at 35 ℃, testing the apparent motility rate, the motility rate and the density of the unfrozen boar semen, and evaluating the data results as shown in table 1.
The method for detecting the sperm motility rate and the acrosome integrity rate comprises the following steps:
(1) the semen immediately after thawing was checked for viability at 35-37 ℃:1 drop of seminal fluid was dropped onto a glass slide and then observed under a 500-fold microscope. And in a microscope visual field, the sperm motility conditions of the upper, middle and lower liquid levels of the semen are comprehensively observed, the sperm motility rate is evaluated, and the process is repeated for 5 times.
(2) Taking a drop of unfrozen semen on a glass slide and preparing into a smear, naturally drying the smear, fixing the smear for 15min by using 2mL of formalin phosphate buffer solution, washing and drying the smear, staining the smear for 90min by using Jiemsa, removing the staining solution, drying the smear in the air, observing 300 sperms under a biological microscope 1000 times, calculating the integrity rate of a plasma membrane, and repeating the steps for 5 times.
TABLE 1
Example 5
The process of example 1 was followed except that: the diluent was prepared as follows: mixing 5g of polyglutamic acid, 0.5g of lactose and 0.05g of beta-galactosidase in 30g of water, reacting at 30 ℃ for 24h and pH 3.5, adding 6g of PVA, continuing the reaction for 20min, adding 5g of glycerol, 30g of glucose, 4g of sodium citrate, 3.2g N-2-hydroxyethylpiperazine-N' -2-ethanesulfonate, 7g of Tris, 4g of EDTA-Na20.1g of cysteine, 50g of a yolk diluent (consisting of water, yolk and spectinomycin in a ratio of 1:1: 0.5) and 70g of water were mixed uniformly to obtain a diluent, and the results are shown in Table 2.
Example 6
The process of example 1 was followed except that: the diluent was prepared as follows: 12g of polyglutamic acid, 8g of lactose and 0.08g of beta-galactosidase are mixed in 30g of water, reacted at 30 ℃ for 24h and pH 3.5, 4g of PVA are added, the reaction is continued for 20min, and then 8g of glycerol, 22g of glucose, 8g of sodium citrate, 2g N-2-hydroxyethylpiperazine-N' -2-ethanesulfonate, 4g of Tris, 1.5g of EDTA-Na20.5g of cysteine, 20g of a yolk diluent (consisting of water, yolk and spectinomycin in a ratio of 1:1: 0.5) and 70g of water were mixed uniformly to obtain a diluent, and the results are shown in Table 2.
Example 7
The process of example 1 was followed except that: the diluent was prepared as follows: mixing 6g of polyglutamic acid, 3g of lactose (weight ratio of lactose to polyglutamic acid is 1:2) and 0.08g of beta-galactosidase in 30g of water, reacting at 30 ℃ for 24h and pH 3.5, adding 4g of PVA, continuing the reaction for 20min, adding 8g of glycerol, 22g of glucose, 8g of sodium citrate, 2g N-2-hydroxyethylpiperazine-N' -2-ethanesulfonic acid, 4g of Tris, 1.5g of EDTA-Na2Mixing 0.5g cysteine, 20g yolk diluent (composed of water, yolk and spectinomycin at a ratio of 1:1: 0.5) and 70g water uniformly to obtain the final productThe results are shown in Table 2.
Example 8
The process of example 1 was followed except that: the diluent was prepared as follows: 10g of polyglutamic acid, 0.6g of lactose and 0.08g of beta-galactosidase were mixed in 30g of water, reacted at 30 ℃ for 24 hours at pH 3.5, 4g of PVA were added and the reaction was continued for 20 minutes, followed by 8g of glycerol, 22g of glucose, 8g of sodium citrate, 2g N-2-hydroxyethylpiperazine-N' -2-ethanesulfonate, 4g of Tris, 1.5g of EDTA-Na20.5g of cysteine, 20g of a yolk diluent (consisting of water, yolk and spectinomycin in a ratio of 1:1: 0.5) and 70g of water were mixed uniformly to obtain a diluent, and the results are shown in Table 2.
Example 9
The process of example 1 was followed except that: the molecular weight of polyglutamic acid is 100 ten thousand (commercially available from Cian-Tonze Biotech Co., Ltd.), as follows:
mixing polyglutamic acid 8g, lactose 2g (weight ratio of lactose to polyglutamic acid is 1:4) and beta-galactosidase 0.04g in water 30g, reacting at 30 deg.C for 24h, pH 3.5, adding PVA 5g, continuing reaction for 20min, adding glycerol 8g, glucose 25g, sodium citrate 4.5g, sodium N-2-hydroxyethyl piperazine-N' -2-ethanesulfonate 2.8g, Tris 5g, and EDTA-Na 2.5g20.2g of cysteine, 30g of a yolk diluent (consisting of water, yolk and spectinomycin in a ratio of 1:1: 0.5) and 70g of water were mixed uniformly to obtain a diluent, and the results are shown in Table 2.
Example 10
The process of example 1 was followed except that: polyglutamic acid has a molecular weight of less than 2 million (commercially available from Cianton Biotech, Inc.), as follows:
mixing 8g of polyglutamic acid, 2g of lactose (the weight ratio of lactose to polyglutamic acid is 1:4) and 0.04g of beta-galactosidase in 30g of water, reacting at 30 ℃ for 24h and pH 3.5, adding 5g of PVA, continuing the reaction for 20min, adding 8g of glycerol, 25g of glucose, 4.5g of sodium citrate, 2.8g of N-2-hydroxyethylpiperazine-N' -2-ethanesulfonic acid sodium salt, 5g of Tris, and sodium acetate, and adding water,2.5g of EDTA-Na20.2g of cysteine, 30g of a yolk diluent (consisting of water, yolk and spectinomycin in a ratio of 1:1: 0.5) and 70g of water were mixed uniformly to obtain a diluent, and the results are shown in Table 2
Example 11
The process of example 1 was followed except that: the egg yolk diluent does not contain spectinomycin, and the egg yolk diluent specifically comprises the following components:
mixing polyglutamic acid 8g, lactose 2g (weight ratio of lactose to polyglutamic acid is 1:4) and beta-galactosidase 0.04g in water 30g, reacting at 30 deg.C for 24h, pH 3.5, adding PVA 5g, continuing reaction for 20min, adding glycerol 8g, glucose 25g, sodium citrate 4.5g, sodium N-2-hydroxyethyl piperazine-N' -2-ethanesulfonate 2.8g, Tris 5g, and EDTA-Na 2.5g20.2g of cysteine, 30g of a yolk diluent (consisting of water, yolk and spectinomycin in a ratio of 1:1: 0.5) and 70g of water were mixed uniformly to obtain a diluent, and the results are shown in Table 2.
Example 12
The process of example 1 was followed except that: the egg yolk diluent does not contain spectinomycin, and the egg yolk diluent specifically comprises the following components:
mixing polyglutamic acid 8g, lactose 2g (weight ratio of lactose to polyglutamic acid is 1:4) and beta-galactosidase 0.04g in water 30g, reacting at 30 deg.C for 24h, pH 3.5, adding PVA 5g, continuing reaction for 20min, adding glycerol 8g, glucose 25g, sodium citrate 4.5g, sodium N-2-hydroxyethyl piperazine-N' -2-ethanesulfonate 2.8g, Tris 5g, and EDTA-Na 2.5g20.2g of cysteine, 30g of a yolk diluent (consisting of water, yolk and spectinomycin in a ratio of 1:1: 0.8) and 70g of water were mixed uniformly to obtain a diluent, and the results are shown in Table 2.
Comparative example 5
The process of example 1 was followed except that: the diluent does not contain polyglutamic acid, and the specific steps are as follows:
mixing 2g lactose and 0.04g beta-galactosidase in 30g water, reacting at 30 deg.C for 24 hr and pH 3.5, adding 5g PVA, reacting for 20min, and adding 8g glycerolOil, 25g glucose, 4.5g sodium citrate, 2.8g sodium N-2-hydroxyethylpiperazine-N' -2-ethanesulfonate, 5g Tris, 2.5g EDTA-Na20.2g of cysteine, 30g of a yolk diluent (consisting of water, yolk and spectinomycin in a ratio of 1:1: 0.8) and 70g of water were mixed uniformly to obtain a diluent, and the results are shown in Table 2.
Comparative example 6
The process of example 1 was followed except that: starch is used to replace lactose, and amylase is used to replace beta-galactosidase, as follows:
mixing 8g of polyglutamic acid, 2g of starch and 0.04g of amylase in 30g of water, reacting at 30 ℃ for 24 hours at pH 3.5, adding 5g of PVA, continuing the reaction for 20min, adding 8g of glycerol, 25g of glucose, 4.5g of sodium citrate, 2.8g of sodium N-2-hydroxyethylpiperazine-N' -2-ethanesulfonate, 5g of Tris, 2.5g of EDTA-Na20.2g of cysteine, 30g of a yolk diluent (consisting of water, yolk and spectinomycin in a ratio of 1:1: 0.8) and 70g of water were mixed uniformly to obtain a diluent, and the results are shown in Table 2.
Comparative example 7
The process of example 1 was followed except that: the diluent contained no PVA and the results are shown in table 2.
TABLE 2
Comparative example 8
Cooling the diluted semen to 0-5 ℃ at the speed of 1-2 ℃/min, then directly putting the diluted semen into liquid nitrogen to cool to-196 ℃, and obtaining the frozen semen.
Comparative example 9
Directly putting the diluted semen into liquid nitrogen to cool to-196 ℃ to obtain the frozen semen.
TABLE 3
The preferred embodiments of the present invention have been described above in detail, but the present invention is not limited thereto. Within the scope of the technical idea of the invention, many simple modifications can be made to the technical solution of the invention, including various technical features being combined in any other suitable way, and these simple modifications and combinations should also be regarded as the disclosure of the invention, and all fall within the scope of the invention.
Claims (10)
1. A preservation method of frozen pig semen is characterized by comprising the following steps:
mixing the pig semen in a pretreatment agent for pretreatment to obtain pretreated semen;
mixing the pre-treated semen with a diluent for dilution to obtain diluted semen;
carrying out program freezing on the diluted semen to obtain frozen semen;
wherein the diluent comprises, based on 100g of water: 5-12g of glycerol, 3-8g of PVA, 5-12g of polyglutamic acid, 0.5-8g of lactose, 0.02-0.08g of beta-galactosidase and 100g of water.
2. A preservation process according to claim 1, wherein the diluent comprises, based on 100g of water: 5-12g of glycerol, 4-6g of PVA, 6-10g of polyglutamic acid, 0.6-3g of lactose, 0.02-0.08g of beta-galactosidase and 100g of water.
3. The preservation method according to claim 1, wherein the diluent further comprises, based on 100g of water: 22-30g of glucose, 4-8g of sodium citrate, 2-3.2g of sodium N-2-hydroxyethylpiperazine-N' -2-ethanesulfonate, 4-7g of Tris and 1.5-4g of EDTA-Na20.1-0.5g of cysteine and 20-50g of egg yolk diluent.
4. A preservation method according to any one of claims 1-3, further comprising the preparation of a diluent, wherein the method of preparing the diluent comprises:
polyglutamic acid, lactose and beta-galactosidase are mixed in water and react for 5-40h at 20-40 ℃ with the pH value of 3-5, and then glucose, sodium citrate, N-2-hydroxyethyl piperazine-N' -2-sodium ethanesulfonate, Tris, EDTA-Na2, cysteine and egg yolk diluent are added and mixed evenly to obtain the diluent.
5. A preservation process according to claim 4, wherein the weight ratio of lactose to polyglutamic acid is 1: 1-10.
6. The preservation method according to claim 4, wherein the molecular weight of the polyglutamic acid is 7 to 10 ten thousand;
preferably, the molecular weight of the PVA is 20000-.
7. A preservation method according to any one of claims 1 to 6, characterized in that the pretreatment agent comprises, based on 100g of water: 2-5g of sodium chloride, 3-8mM of amino acid, 0.1-0.5g of creatine phosphate and 100g of water.
8. A preservation process according to claim 7, wherein the pre-treatment comprises: and (3) standing the boar semen in the pretreating agent for 0.2-2h at the temperature of 18-22 ℃, then centrifuging, and removing a supernatant to obtain the pretreated semen.
9. The preservation method according to claim 8, wherein the volume ratio of the porcine semen to the pretreatment agent is 1: 3-8.
10. A preservation method according to any one of claims 1 to 9, wherein the method of program freezing comprises: cooling the diluted semen to 0-5 ℃ at the speed of 1-2 ℃/min, preserving the heat for 10-30min, and then putting the diluted semen into liquid nitrogen to cool to-196 ℃.
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