CN106508886B - Lycopene and application of the alpha-lipoic acid as mammal semen cryopreservation agent - Google Patents
Lycopene and application of the alpha-lipoic acid as mammal semen cryopreservation agent Download PDFInfo
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- CN106508886B CN106508886B CN201610825636.9A CN201610825636A CN106508886B CN 106508886 B CN106508886 B CN 106508886B CN 201610825636 A CN201610825636 A CN 201610825636A CN 106508886 B CN106508886 B CN 106508886B
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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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Abstract
The present invention relates to lycopenes and application of the alpha-lipoic acid compatibility as mammal semen cryopreservation liquid.Present invention employs lycopenes and alpha-lipoic acid, and the physiological activity of mammalian sperm can be protected well during the freeze-thaw of mammal sperm.
Description
Technical field
The present invention relates to mammal semen cryopreservation agent, and in particular to lycopene and alpha-lipoic acid compatibility are as the food in one's mouth
The application of newborn animal semen Cryoprotectant.
Background technique
The freezen protective technology of mammal sperm is widely used in animal productiong, and it is efficiently numerous to become excellent domestic animal kind
Educate, accelerate the important means of crossbreeding and improvement；It also provides not for animal embryo transplanting, in vitro fertilization etc. by time and space simultaneously
The material of limitation.But generally existing physical injury, chemical lesion and oxygen to sperm during freezing of semen at present
Change stress damage etc., greatly reduces semen cryopreservation effect.In the process of cryopreservation of sperm, it is necessary to dilute freezing
The anti-oxidant protective agent that external source is added in liquid is released, to reduce ROS to the oxidative damage of plasmalemmae of sperms.
Summary of the invention
The object of the present invention is to provide a kind of dilutions for the low damage freezen protective of mammalian sperm.
Component of the present invention using lycopene and alpha-lipoic acid as the freeze-extender of mammal sperm is protected with freezing
Deposit the antioxidases such as rear goat sperm motility rate, plasm membrane integrity, acrosomal integrity, mitochondria activity and SOD, CAT, GSH-Px
Activity etc. is used as deliberated index, and probes into the best compatibility concentration of lycopene and alpha-lipoic acid, to keep mammalian sperm
There is good physiological activity after freeze-thaw.
The present invention additionally provides the freeze-extender of the mammal sperm comprising lycopene and alpha-lipoic acid simultaneously.
Further, the freeze-extender component of mammal sperm of the invention are as follows: mammal sperm described in every 100mL
Freeze-extender formula are as follows: Tris 3.25g, citric acid 1.74g, fructose 1.14g, penicillin 200000IU, streptomysin
100000IU, yolk 15mL, glycerol 5mL, lycopene 100mg, alpha-lipoic acid 0.5mg, remaining is water.
In another scheme, the freeze-extender component of mammal sperm of the invention are as follows: lactation described in every 100mL is dynamic
The formula of the freeze-extender of object sperm are as follows: Tris 3.25g, citric acid 1.74g, fructose 1.14g, penicillin 200000IU, chain
Mycin 100000IU, yolk 15mL, lycopene 100mg, alpha-lipoic acid 0.5mg, remaining is water.
The chill back formula of liquid of mammal sperm of the invention, due to using lycopene and alpha-lipoic acid, energy
Enough physiological activity for protecting mammalian sperm well during the freeze-thaw of mammal sperm.
Detailed description of the invention
Fig. 1 is SOD (A figure), CAT (B figure) and GSH-Px (C after lycopene and alpha-lipoic acid compatibility freeze goat sperm
Figure) active influence.
Specific embodiment
Below in conjunction with specific embodiment, the present invention is further described in detail.
Embodiment:
Technical solution according to the invention, the present embodiment provide six groups of tomato reds for mammal semen cryopreservation
The dilution formula of liquid of element and alpha-lipoic acid compatibility, weighs 0mg, 50mg, 100mg lycopene and 0mg, 0.5mg, 1mg α-respectively
Lipoic acid, according to carrying out compatibility shown in table 1, other compositions weigh the two according to ratio of the table 2 shared by it.
1 lycopene of table and alpha-lipoic acid compatibility program
2 lycopene of table and alpha-lipoic acid compatibility dilution formula of liquid (every 100ml)
Note: mycillin is dual anti-individually to be added before cryoprotection.
The freezen protective dilution of mammal sperm is used for the freeze-thaw of mammal sperm in above-described embodiment
Method follows these steps to implement:
(1) after detection is qualified, according to sperm: semen dilution is added containing difference the ratio that chill back I liquid is 1:2
The chill back I liquid of concentration lycopene and alpha-lipoic acid is placed in cooling balance 2h in 4 DEG C of refrigerators；
(2) the chill back II liquid of isothermal (4 DEG C) is added according to the ratio of 1:2 for the sperm after first time being diluted and balanced
It further dilutes, balances 1.5h in 4 DEG C of refrigerators；
(3) -5 DEG C are down to from 5 DEG C with the rate of 1 DEG C/min, tubule is placed in above liquid nitrogen surface at 2~3cm up to 6~
Then tubule is put into freezen protective in liquid nitrogen by 8min；
(4) after freezing 14d, thaw 30s in 37 DEG C of water-baths, and sperm motility rate, acrosomal integrity, plasma membrane are complete after measurement is frozen
The activity value of whole rate, mitochondria activity and antioxidase (SOD, CAT, GSH-Px).
Lycopene and alpha-lipoic acid are added according to 2 compatibility program of table, under same formula, other reagents in addition to glycerol
Mixed liquor be chill back I liquid, the mixed liquor of all reagents (including glycerol) is chill back II liquid.
Test freezing goat sperm used comes from Yulin Northern Shaanxi White Cashmere Goat proving ground.Kind is acquired using Pseudopyloric metaplasia
Ram sperm carries out Ordinary fruit quality inspection, and sperm motility, which reaches 0.8 or more, can be used for freezen protective, transport experiment back in lh
Room.
Chill back I liquid is added according to the ratio of 1:2 in sperm after will test qualification, after multilayer yarn cloth package, places
The cooling balance 2h in 4 DEG C of refrigerators.Then the sperm after first time being diluted and balanced is added (the 4 of isothermal according to the ratio of 1:2
DEG C) chill back II liquid further dilute, make the final ratio 1:4 of sperm and dilution, through multilayer yarn cloth package after, 4
1.5h is balanced in DEG C refrigerator.After tubulature, tubule is put into Slow-rate freezing instrument control its be down to from 5 DEG C with the rate of 1 DEG C/min-
5 DEG C, then place it in 2~3cm above liquid nitrogen surface and fumigate 6~8min, tubule is finally put into freezen protective in liquid nitrogen.
Srozen tube is put into 37 DEG C of water-baths the 30s that thaws after preservation 14d.Under normal temperature environment, in 400 × optical microscopy
Lower observation simultaneously counts the sperm count linearly moved, calculates sperm motility rate (table 3).
Perforatorium is detected using the method for the fluorescein-labeled peanut agglutinin of isothiocyanates (FITC-PNA) dyeing
30 μ L semen samples are uniformly applied on glass slide by percentage of head rice, after spontaneously drying, then the fixed 10min of methanol is added
10 μ L FITC-PNA dye liquors, are protected from light under 37 DEG C of wet environments and are incubated for 30min, and PBS is rinsed 2 times, is air-dried, with colourless nail
Sheet for oil seal in fluorescence microscopy microscopic observation and calculates Sperm acrasomal integvity (table 3).
Plasmalemmae of sperms percentage of head rice is detected using hypo-osmotic swelling test (HOST), that is, the semen sample after taking 20 μ L defrosting to be incubated for
200 μ L of isothermal HOST hypotonic solution is added to mix, takes 10 μ L to be uniformly applied on glass slide after 37 DEG C of incubations 1h, 400 ×
5 visuals field of microscopically observation are set, the curved tail rate (table 3) of sperm is calculated.
1 μ L propidium iodide (PI) is stored liquid and 1 μ L Rhodamine 123 (Rh123) is store by the active detection of mitochondria of sperms
It is protected from light culture 10min after liquid storage mixing, the semen dilution I liquid of the sperm after defrosting is diluted, draws 50 μ L and above-mentioned be protected from light is added
PI the and Rh123 mixed liquor of culture cultivates 30min, takes 10 μ L sample film-makings, then observe simultaneously under 400 × inverted microscope
It is active (table 3) to calculate mitochondria of sperms.
Kit specification provided by biotechnology company is built up to superoxide dismutase in sperm according to Nanjing
(SOD), the activity of the enzymes such as catalase (CAT) and glutathione peroxidase (GSH-Px) is detected (Fig. 1), and will
Data measured substitutes into correlation formula and accurately calculates, and all results are indicated with mean+SD.
The influence of table 3 joint addition lycopene and alpha-lipoic acid to goat sperm refrigerating effect
According to the above operating procedure, it is complete to measure goat sperm motility rate, acrosomal integrity, plasma membrane after obtaining freezing 14d
Rate, mitochondria activity and antioxidase (SOD, CAT, GSH-Px) activity.
It should be understood that for those of ordinary skills, can improvement or transformation based on the above description, and
All these modifications and variations should all belong to the protection domain of appended claims of the present invention.
Claims (1)
1. a kind of freeze-extender of goat sperm, which is characterized in that the freeze-extender component of the goat sperm are as follows: every
The formula of the freeze-extender of mammal sperm described in 100mL are as follows: Tris 3.25g, citric acid 1.74g, fructose 1.14g are green
Mycin 200000IU, streptomysin 100000IU, yolk 15mL, glycerol 5mL, lycopene 100mg, alpha-lipoic acid 0.5mg, remaining
For water.
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| CN201610825636.9A CN106508886B (en) | 2016-09-14 | 2016-09-14 | Lycopene and application of the alpha-lipoic acid as mammal semen cryopreservation agent |
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| CN201610825636.9A CN106508886B (en) | 2016-09-14 | 2016-09-14 | Lycopene and application of the alpha-lipoic acid as mammal semen cryopreservation agent |
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| CN106508886B true CN106508886B (en) | 2019-11-12 |
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Citations (5)
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| CN103004750A (en) * | 2012-11-27 | 2013-04-03 | 云南省畜牧兽医科学院 | Livestock semen freezing diluent as well as preparation method and application thereof |
| CN104161036A (en) * | 2014-07-15 | 2014-11-26 | 西北农林科技大学 | Antifreeze agent and diluent for livestock semen refrigeration preservation and application thereof |
| CN104542577A (en) * | 2015-02-01 | 2015-04-29 | 吉林大学 | Application of lycopene in preparation of human semen cryoprotectant |
| CN104705290A (en) * | 2015-03-19 | 2015-06-17 | 新疆天山畜牧生物工程股份有限公司 | Sheep frozen semen dilution and process used for preparing sheep straw frozen semens |
| CN105638643A (en) * | 2016-02-16 | 2016-06-08 | 云南省畜牧兽医科学院 | Freezing diluent for seminal fluid of livestock |
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2016
- 2016-09-14 CN CN201610825636.9A patent/CN106508886B/en active Active
Patent Citations (5)
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|---|---|---|---|---|
| CN103004750A (en) * | 2012-11-27 | 2013-04-03 | 云南省畜牧兽医科学院 | Livestock semen freezing diluent as well as preparation method and application thereof |
| CN104161036A (en) * | 2014-07-15 | 2014-11-26 | 西北农林科技大学 | Antifreeze agent and diluent for livestock semen refrigeration preservation and application thereof |
| CN104542577A (en) * | 2015-02-01 | 2015-04-29 | 吉林大学 | Application of lycopene in preparation of human semen cryoprotectant |
| CN104705290A (en) * | 2015-03-19 | 2015-06-17 | 新疆天山畜牧生物工程股份有限公司 | Sheep frozen semen dilution and process used for preparing sheep straw frozen semens |
| CN105638643A (en) * | 2016-02-16 | 2016-06-08 | 云南省畜牧兽医科学院 | Freezing diluent for seminal fluid of livestock |
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| α-硫辛酸、维生素P和L-肉碱对猪精液常温和低温保存效果的研究;刘亚伟;《中国优秀硕士学位论文全文数据库农业科技缉》;20150215(第2期);第16页 * |
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