CN108812646A - A kind of stored refrigerated dilution of dog sperm - Google Patents
A kind of stored refrigerated dilution of dog sperm Download PDFInfo
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- CN108812646A CN108812646A CN201810863586.2A CN201810863586A CN108812646A CN 108812646 A CN108812646 A CN 108812646A CN 201810863586 A CN201810863586 A CN 201810863586A CN 108812646 A CN108812646 A CN 108812646A
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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
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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
- A01N1/0226—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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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/81—Solanaceae (Potato family), e.g. tobacco, nightshade, tomato, belladonna, capsicum or jimsonweed
- A61K36/815—Lycium (desert-thorn)
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P39/00—General protective or antinoxious agents
- A61P39/06—Free radical scavengers or antioxidants
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Abstract
A kind of stored refrigerated dilution of dog sperm, is made of formula as below：A liquid：5-10 parts of lycium ruthenicum extract, 5-10 parts of L-BETAIN, 2-5 parts of L-arginine, 3-7 parts of epiphysin；B liquid：3-5 parts of vitamin E, 5-10 parts of glucose, 10-15 parts of citric acid, 20-40 parts of g of fructose, 20-40 parts of soybean lecithin, ten thousand IU/100ml of penicillin 30-40, ten thousand IU/100ml of streptomysin 20-50,10-20 parts of trehalose, 10-15 parts of peptone, 5-10 parts of hyaluronic acid, and is allowed using special extraction process and the motility rate after sperm refrigeration, the percentage of head rice of acrosome after refrigeration are obviously improved using the dilution of inventive formulation.
Description
Technical field
The invention belongs to technical field of animal reproduction, and in particular to a kind of stored refrigerated dilution of dog sperm.
Background technique
Refrigeration is made by the processing of the relevant technologies in today of biotechnology rapid development, the sperm of some outstanding sires
Sperm, and vigorously promoted the use by technology of artificial insemination.Wherein the combination of dog cryopreserved semen and technology of artificial insemination is in the modern times
There are boundless market prospects in canine farming, firstly, it can substantially reduce the infection of genital system diseases；Secondly, it can
So that good species public affairs dog is fully used, breed improvement is not only improved, and the feeding cost of public dog can be saved；Furthermore due to
Cryopreserved semen can save, long distance transportation, thus, artificial insemination can overcome due to epoch, geographical separation and male and female dog body
It breeds caused by lattice difference difficult.Although the stored refrigerated research of the sperm of dog has been carried out many years, when sperm is survived
Between it is shorter, Semen routine effect is poor, and conception rate and litter size are substantially less than fresh semen after artificial insemination.Dilution is sperm
Essential constituent when refrigeration plays the role of dilution, buffering, nutrition and protection to sperm in cold storage procedure, and
Influence of the low temperature to sperm can be effectively reduced, there are some researches prove the constituent by changing semen diluent can drop
Damage of the low cold storage procedure to sperm.
Lycium ruthenicum contains a large amount of procyanidine, is so far, to find the highest natural open country of procyanidine (OPC) content
Plant, OPC are most effective water-soluble free radical scavengers, have very strong anti-aging effects, are a kind of novel height
Imitate antioxidant.It is demonstrated experimentally that the free radical resisting oxidability of OPC is 50 times of vitamin E, ascorbic 20 times, and absorb
Rapidly completely, it can reach highest haemoconcentration within oral 20 minutes, metabolic half life was up to 7 hours；Due to more on dog plasmalemmae of sperms
Unsaturated fatty acid content is abundant, when encountering active chalcogen (ROS) attack, peroxidatic reaction of lipid easily occurs, causes sperm
Oxidative stress loses fertility.Under normal circumstances, contain antioxidant in refining, be enough that ROS is maintained to generate and remove it
Between balance, but during semen dilution saves, antioxidant concentration is reduced in refining, and balance is broken, and ROS can not
It is timely and effectively removed, sperm is made to generate oxidative damage.Therefore, it needs to add during semen dilution saves suitable anti-
Oxidant enhances the oxidation resistance of sperm, as far as possible damage of the reduction ROS to dog-like sperm.On the other hand, antioxidant L- meat
Malicious alkali and antioxidant L-arginine can promote transhipment of the fatty acid of activation from cytoplasm to mitochondrial matrix, reduce ROS and produce
Raw and peroxidatic reaction of lipid generation；Epiphysin (Melatonin, MLT) be it is a kind of synthesized and secreted by pineal body it is important
Amine bormones has oxidation resistance, can protect the ingredients such as the Cell membrane lipids, nucleic acid, protein of cell.
Summary of the invention
The purpose of the present invention is to solve the defect problems of the above-mentioned prior art, and it is stored refrigerated dilute to provide a kind of dog sperm
Release liquid.
The technical scheme is that：The stored refrigerated dilution mixed according to parts by weight by following raw material proportioning and
At：A liquid：5-10 parts of lycium ruthenicum extract, 5-10 parts of L-BETAIN, 2-5 parts of L-arginine, 3-7 parts of epiphysin；B liquid：Vitamin
E3-5 parts, 5-10 parts of glucose, 10-15 parts of citric acid, 20-40 parts of g of fructose, 20-40 parts of soybean lecithin, penicillin 30-40
Ten thousand IU/100ml, ten thousand IU/100ml of streptomysin 20-50,10-20 parts of trehalose, 10-15 parts of peptone, 5-10 parts of hyaluronic acid.
A kind of stored refrigerated dilution of dog sperm：The stored refrigerated dilution by following raw material proportioning according to parts by weight
It mixes：A liquid：8 parts of lycium ruthenicum extract, 7 parts of L-BETAIN, 3 parts of L-arginine, 4 parts of epiphysin；B liquid：Vitamin E 4
Part, 7 parts of glucose, 12 parts of citric acid, 30 parts of g of fructose, 30 parts of soybean lecithin, 350,000 IU/100ml of penicillin, streptomysin 35
Ten thousand IU/100ml, 15 parts of trehalose, 12 parts of peptone, 7 parts of hyaluronic acid.The lycium ruthenicum extract extracting mode is：
(1) feedstock processing：Lycium ruthenicum impurity elimination again is taken according to parts by weight, is cleaned, and the freeze-day with constant temperature that temperature is 65 DEG C is placed in
Drying for 24 hours, smashes it through 0.2mm sieve in case；
(2) ultrasonic wave added is handled:Coarse powder made from step (1) is added into Ultrasound Instrument, adds the dense of 6 times of weight equivalents
Degree be 65% ethyl alcohol, control ultrasonic power be 350W, ultrasonic time 100min；
(3) ultra high pressure treatment：Medical fluid in step (2) after ultrasound is transferred in intermittent ultra high pressure extraction machine, emptying receptacles,
500MPa is boosted to, 5min is kept；
(4) macroporous resin adsorption purifies：HIGH PRESSURE TREATMENT liquid obtained in step (3), being placed in specification is 1000mm × 100mm
DL08 model highly acidic cation-D301R model weakly-basic anion-DL08 model strong acidic ion resin adsorbed
Separation, control adsorption temp are 30-40 DEG C, and flow control 0.5-1ml/min obtains extracting solution.
The preparation method of the stored refrigerated dilution of dog sperm is：Raw material in B liquid is proportionally mixed in sterile
It is dissolved in beaker in 100ml distilled water, adjusts PH6.8, stirred 6min, gained A liquid is proportionally mixed and is incorporated in sterile burning
It is dissolved in 100ml distilled water in cup, mixes A, B liquid, adjusts PH7.0 after stirring 5min, is placed in preservation in 2 DEG C of refrigerating boxes.
Beneficial effects of the present invention：
(1) there is the good active effect of enhancing to canine semen, enhance its resisting stress, especially have to inoxidizability
Stronger effect, can effectively less refrigeration damage, raising living spermatozoa percentage and acrosomal integrity；
(2) high using effective component procyanidin content in the lycium ruthenicum extract of purpose made technology preparation, purity is high.
Specific embodiment
Embodiment 1
Stored refrigerated dilution is mixed according to parts by weight by following raw material proportioning：A liquid：8 parts of lycium ruthenicum extract,
7 parts of L-BETAIN, 3 parts of L-arginine, 4 parts of epiphysin；B liquid：4 parts of vitamin E, 7 parts of glucose, 12 parts of citric acid, fructose 30
Part g, 30 parts of soybean lecithin, 350,000 IU/100ml of penicillin, 350,000 IU/100ml of streptomysin, 15 parts of trehalose, peptone 12
Part, 7 parts of hyaluronic acid.
The lycium ruthenicum extract extracting mode is：
(1) feedstock processing：Lycium ruthenicum impurity elimination again is taken according to parts by weight, is cleaned, and the freeze-day with constant temperature that temperature is 65 DEG C is placed in
Drying for 24 hours, smashes it through 0.2mm sieve in case；
(2) ultrasonic wave added is handled:Coarse powder made from step (1) is added into Ultrasound Instrument, adds the dense of 6 times of weight equivalents
Degree be 65% ethyl alcohol, control ultrasonic power be 350W, ultrasonic time 100min；
(3) ultra high pressure treatment：Medical fluid in step (2) after ultrasound is transferred in intermittent ultra high pressure extraction machine, emptying receptacles,
500MPa is boosted to, 5min is kept；
(4) macroporous resin adsorption purifies：HIGH PRESSURE TREATMENT liquid obtained in step (3), being placed in specification is 1000mm × 100mm
DL08 model highly acidic cation-D301R model weakly-basic anion-DL08 model strong acidic ion resin adsorbed
Separation, control adsorption temp are 30-40 DEG C, and flow control 0.5-1ml/min obtains extracting solution.
Raw material in B liquid is proportionally mixed in sterile beaker and is dissolved in 100ml distilled water, PH6.8, stirring are adjusted
6min, gained A liquid is proportionally mixed to be incorporated in sterile beaker is dissolved in 100ml distilled water, mixes A, B liquid, stirs 5min
After adjust PH7.0, be placed in preservation in 2 DEG C of refrigerating boxes.
Embodiment 2
Stored refrigerated dilution is mixed according to parts by weight by following raw material proportioning：A liquid：5 parts of lycium ruthenicum extract,
5 parts of L-BETAIN, 2 parts of L-arginine, 3 parts of epiphysin；B liquid：3 parts of vitamin E, 5 parts of glucose, 10 parts of citric acid, fructose 20
Part g, 20 parts of soybean lecithin, 300,000 IU/100ml of penicillin, 200,000 IU/100ml of streptomysin, 10 parts of trehalose, peptone 10
Part, 5 parts of hyaluronic acid.Remaining is the same as embodiment 1.
Embodiment 3
Stored refrigerated dilution is mixed according to parts by weight by following raw material proportioning：A liquid：Lycium ruthenicum extract 10
Part, 10 parts of L-BETAIN, 5 parts of L-arginine, 7 parts of epiphysin；B liquid：5 parts of vitamin E, 10 parts of glucose, 15 parts of citric acid, fruit
40 parts of g of sugar, 40 parts of soybean lecithin, 400,000 IU/100ml of penicillin, 500,000 IU/100ml of streptomysin, 20 parts of trehalose, albumen
15 parts of peptone, 10 parts of hyaluronic acid.Remaining is the same as embodiment 1.
Embodiment 4
Stored refrigerated dilution is mixed according to parts by weight by following raw material proportioning：A liquid：2 parts of lycium ruthenicum extract,
2 parts of L-BETAIN, 1 part of L-arginine, 5 parts of epiphysin；B liquid：Vitamin E2 part, 2 parts of glucose, 2 parts of citric acid, 7 parts of fructose
G, 20 parts of soybean lecithin, 100,000 IU/100ml of penicillin, 100,000 IU/100ml of streptomysin, 5 parts of trehalose, 5 parts of peptone, thoroughly
Bright 5 parts of matter acid.Remaining is the same as embodiment 1.
Comparative example 1
Be not added with A liquid remaining with embodiment 1.
Comparative example 2
The extracting mode of lycium ruthenicum extract is conventional boiling：5 parts of water of dosing material weight impregnate 50 minutes, add 6 times of weights
The water of amount decocts three times, and 2 hours every time, collecting decoction；Remaining is the same as embodiment 1.
Above-described embodiment 1-4, comparative example 1-2 are subjected to the refrigeration (4 DEG C) of sperm, observation refrigerates motility rate variation in 5 days, preceding
Sperm motility rate and acrosomal integrity afterwards the results are shown in Table one, table two.
One sperm of table refrigerates motility rate variation in 5 days
Two perforatorium acrosomal integrity of table
The sperm motility rate of the embodiment 1-3 within inventive formulation technique, perforatorium are complete as can be seen from the above table
Rate solution refrigeration after it is higher, especially be with embodiment 1 it is best, refrigerate the 5th day after motility rate reach 0.65 ± 0.01, acrosomal integrity
Reach 0.76 ± 0.01, the A formula function and effect for illustrating that the present invention uses are significant, the embodiment except inventive formulation range
4, effect is far short of what is expected, and 1 effect of comparative example for being not added with A liquid is worst, in conjunction with comparative example 2 and embodiment 1 it can be seen that the present invention
The extraction process of use has a certain upgrade effect to the survival rate of sperm, the percentage of head rice of acrosome.
Claims (4)
1. a kind of stored refrigerated dilution of dog sperm, it is characterised in that：The stored refrigerated dilution is pressed by following raw material proportioning
It is mixed according to parts by weight：A liquid：5-10 parts of lycium ruthenicum extract, 5-10 parts of L-BETAIN, 2-5 parts of L-arginine, epiphysin
3-7 parts；B liquid：3-5 parts of vitamin E, 5-10 parts of glucose, 10-15 parts of citric acid, 20-40 parts of g of fructose, soybean lecithin 20-
40 parts, ten thousand IU/100ml of penicillin 30-40, ten thousand IU/100ml of streptomysin 20-50,10-20 parts of trehalose, 10-15 parts of peptone,
5-10 parts of hyaluronic acid.
2. a kind of stored refrigerated dilution of dog sperm, it is characterised in that：The stored refrigerated dilution is pressed by following raw material proportioning
It is mixed according to parts by weight：A liquid：8 parts of lycium ruthenicum extract, 7 parts of L-BETAIN, 3 parts of L-arginine, 4 parts of epiphysin；B liquid：
4 parts of vitamin E, 7 parts of glucose, 12 parts of citric acid, 30 parts of g of fructose, 30 parts of soybean lecithin, 350,000 IU/100ml of penicillin,
350,000 IU/100ml of streptomysin, 15 parts of trehalose, 12 parts of peptone, 7 parts of hyaluronic acid.
3. the stored refrigerated dilution of a kind of dog sperm according to claim 2, it is characterised in that：The lycium ruthenicum extract
Extracting mode is：
(1) feedstock processing：Lycium ruthenicum impurity elimination again is taken according to parts by weight, is cleaned, and is placed in the thermostatic drying chamber that temperature is 65 DEG C
Drying for 24 hours, smashes it through 0.2mm sieve；
(2) ultrasonic wave added is handled:Coarse powder made from step (1) is added into Ultrasound Instrument, the concentration for adding 6 times of weight equivalents is
65% ethyl alcohol, control ultrasonic power be 350W, ultrasonic time 100min；
(3) ultra high pressure treatment：Medical fluid in step (2) after ultrasound is transferred in intermittent ultra high pressure extraction machine, emptying receptacles, boosting
To 500MPa, 5min is kept；
(4) macroporous resin adsorption purifies：HIGH PRESSURE TREATMENT liquid obtained in step (3), being placed in specification is 1000mm × 100mm's
DL08 model highly acidic cation-D301R model weakly-basic anion-DL08 model strong acidic ion resin carries out absorption point
From control adsorption temp is 30-40 DEG C, and flow control 0.5-1ml/min obtains extracting solution.
4. the stored refrigerated dilution of a kind of dog sperm according to claim 2, it is characterised in that：The dog sperm refrigeration is protected
The preparation method for depositing dilution is：Raw material in B liquid is proportionally mixed in sterile beaker and is dissolved in 100ml distilled water,
PH6.8 is adjusted, 6min is stirred, gained A liquid proportionally mix to be incorporated in sterile beaker is dissolved in 100ml distilled water, mixing
A, B liquid adjusts PH7.0 after stirring 5min, is placed in preservation in 4 DEG C of refrigerating boxes.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201810863586.2A CN108812646A (en) | 2018-08-01 | 2018-08-01 | A kind of stored refrigerated dilution of dog sperm |
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| CN201810863586.2A CN108812646A (en) | 2018-08-01 | 2018-08-01 | A kind of stored refrigerated dilution of dog sperm |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110547291A (en) * | 2019-09-27 | 2019-12-10 | 安徽医科大学 | efficient antioxidant human oocyte cryoprotectant |
| CN115119828A (en) * | 2022-07-25 | 2022-09-30 | 广州医药研究总院有限公司 | Diluent for refrigerated preservation of canine sperms and preparation method and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110547291A (en) * | 2019-09-27 | 2019-12-10 | 安徽医科大学 | efficient antioxidant human oocyte cryoprotectant |
| CN115119828A (en) * | 2022-07-25 | 2022-09-30 | 广州医药研究总院有限公司 | Diluent for refrigerated preservation of canine sperms and preparation method and application thereof |
| CN115119828B (en) * | 2022-07-25 | 2023-03-07 | 广州医药研究总院有限公司 | Diluent for refrigerating and preserving canine sperms and preparation method and application thereof |
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Application publication date: 20181116 |