CN104542577A - Application of lycopene in preparation of human semen cryoprotectant - Google Patents
Application of lycopene in preparation of human semen cryoprotectant Download PDFInfo
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Abstract
The invention relates to an application of lycopene in preparation of a human semen cryoprotectant, belonging to the field of human semen cryoprotectants. The invention discloses an application of lycopene in the preparation of the human semen cryoprotectant. Every 100mL of the lycopene-containing human semen cryoprotectant comprises the following materials: 1.5g of glucose, 1.3g of trisodium citrate dehydrate, 20ml of glycerin, 4.296*10<-4>-4.296*10<-3>g of lycopene, 20ml of yolk, 0.01-0.1mL of dimethyl sulfoxide and the balance of double distilled water. A certain amount of lycopene is added into the cryoprotectant, so that the human semen cryopreservation effect is obviously improved, the functional parameters such as forward activity ratio and survival rate, of the thawed human semen are obviously higher than those in a control group in which the lycopene is not added, the normal morphology rate, DNA integrity rate and mitochondrial membrane potential are obviously higher than those in the control group, and the sperm apoptosis rate is obviously lower than that in the control group, so that the lycopene has high safety on human semen quality protection.
Description
Technical field
The invention belongs to Seminal plasma cryoprotector field.
Background technology
Sperm is the special cells with genetic material.Seminal fluid is through technical finesse; under the protection of cryoprotector; through certain cooling process; under being kept at ultra low temperature state (in liquid nitrogen); sperm presents hyaloid freezing, and there will not be protoplasm to dewater, and its original structure is protected; so the sperm after thawing still can be recovered, rejuvenate.The sperm metabolism activity of freezing state is subject to obvious suppression, and its life remains static, thus reaches the object that can preserve for a long time.
Sperm freezing protecting agent is the most important material of sperm freezing technology.Sperm cryopreservation agent the most frequently used is at present the compound freezing liquid containing glycerine and yolk, is made up of glycerine, yolk, glucose, sodium citrate etc.Cryoprotector is divided into permeability cryoprotection and impermeability cryoprotection according to the mechanism of action, and glycerine belongs to permeability cryoprotector, and impermeability cryoprotector has fructose, sucrose, yolk, albumin etc.Permeability cryoprotector infiltrates in cell, reduces the difference of intraor extracellular osmotic pressure, when extracellular freezing, alleviates degree and the speed of cell shrinkage; Can also be combined with ICW and electrolyte, cause the stable of Hydrated structure albumen, play freeze proof effect.Impermeability cryoprotector can not enter cell, shields, promotes that ICW is excessive, cell generation shrinkage time freezing, thus reduce the formation of intracellular ice crystal by maintaining the stable of cell membrane.Research shows, in seminal fluid frozen-thaw process, produce a large amount of active oxygen (reactiveoxygenspecies, ROS), the lipid content of plasmalemmae of sperms accounts for more than 50%.And plasmalemmae of sperms is because be rich in polyunsaturated fatty acids (poly-unsaturatedfattyacid, PUFA), wherein, the main component of lipid is phosphatide and cholesterol.Cell membrane is the bilayer structure be made up of lipid, and film lipid has hydrophilic and hydrophobic two parts, is fatty acid.Fatty acid is subject to ROS damage, thus after causing freeze thawing, sperm motility parameters obviously declines.The further investigation that active oxygen has an impact to sperm along with oxidative stress to the damage of sperm, finds that active oxygen produces the impact of every aspect to sperm, mainly contains the following aspects.
1. sperm membrane damage: large quantity research shows the attack being very easily subject to ROS in sperm membrane containing a large amount of polyunsaturated fatty acids at present.Therefore active oxygen causes the minimizing of film polyunsaturated fatty acid to cause the mobility of sperm and permeability to change, and causes the motility of sperm and integrality that irreversible damage occurs.Meanwhile, active oxygen also can cause the damage of mitochondrial membrane, and mitochondrial respiratory function is changed, and the energy that mitochondria of sperms produces reduces, and the locomitivity of sperm reduces.
2. DNA damage: DNA is as the transmitter of genetic material, and its damage directly affects the generation of sperm quality and fertilized egg.Oxidative stress can cause sperm DNA generation strand or two chain break by the generation of free radical, and simultaneously also due to the DNA that mitochondrial DNA is exposed, the protection and the reparation lesion capability itself that lack protein are more weak.Usually cause sperm coup injury, in addition.Active oxygen and metabolite thereof also can produce toxicity to cell, cause Apoptosis, directly cause Sperm lesion.
3. Protein Damage: because active oxygen can the peroxidating of induced protein mercapto can make the function and structure of sperm change, sperm is more easily sustained damage, in addition the generation of a large amount of active oxygen also can cause the damage of acrosome structure, thus reduces the fertility of sperm.In addition, the change that sperm in vitro is cultivated and in ART, the cryoprotection of sperm all can affect the function and structure of sperm, causes a series of changes such as sperm membrane lipid peroxidation, DNA and injury of mitochondria.
Lycopene 1ycopene is acyclic unsaturated carotenoid, belong to the carotenoid (only having carbon hydrogen element to form) in carotenoid, in carotenoid, show stronger non-oxidizability there is scavenging free radicals, promote the different physiological roles such as GJIC, anti-cancer tumor suppressor, its distinctive Composition and function paid close attention to by common people, causes the global research boom to lycopene production technology and effect thereof.Lycopene has been proved to be very effective singlet oxygen quencher, simultaneously also to NO free radical (NO), and sulfonyl (RSO
2) etc. free radical have eliminating effect.
Lycopene is the one of naturally occurring carotenoid in tomato, and carotenoid is constituent important in human body Antioxidative Defense System, and it plays the part of more and more important role in the systems such as internal secretion.In carotenoid, lycopene is not only and commonly also often can be eaten in fruit and food, and it can the most effectively remove singlet, also can remove peroxy radical simultaneously.
Summary of the invention
The invention provides a kind of lycopene and preparing the application in Seminal plasma cryoprotector, to solve the following problem that the compound freezing liquid of sperm containing glycerine and yolk conventional at present exists: Cryopreservation Sperm progressive motility rate is low, anabiosis rate is low, normal morphology rate is low, DNA percentage of head rice is low, apoptosis rate is high.
The technical scheme that the present invention takes is: lycopene is preparing the application in Seminal plasma cryoprotector.
Lycopene is preparing the application that can improve in the Seminal plasma cryoprotector of Cryopreservation Sperm progressive motility rate.
Lycopene is preparing the application that can improve in the Seminal plasma cryoprotector of Cryopreservation sperm anabiosis rate.
Lycopene is preparing the application that can improve in the Seminal plasma cryoprotector of Cryopreservation sperm normal morphology rate.
Lycopene is preparing the application that can improve in the Seminal plasma cryoprotector of Cryopreservation sperm DNA percentage of head rice.
Lycopene is preparing the application that can reduce in the Seminal plasma cryoprotector of Cryopreservation sperm apoptosis rate.
Following material is contained containing in every 100mL Seminal plasma cryoprotector of lycopene:
Glucose 1.5g, trisodium citrate two water 1.3g, glycerine 20ml, lycopene 4.296 × 10
-4g-4.296 × 10
-3g, yolk 20ml, dimethyl sulfoxide (DMSO) 0.01mL-0.1mL, surplus is distilled water.
The preparation method of the Seminal plasma cryoprotector of described lycopene comprises the following steps:
(1) preparation of 99.9mL-99.99mL yolk, the compound cryoprotector of glycerine
(1) weigh 1.5g glucose and 1.3g trisodium citrate two water, add distilled water to 59.9mL-59.99mL;
(2) mix completely after adding 20mL glycerine, filter with 0.45 μm of millipore filter after dissolving completely;
(3) add the fresh yolk of 20mL, make suspension;
(4) to be put by made suspension in 56 DEG C of water-baths 30 minutes, concussion is stirred;
(5) detect the pH value of solution, make pH value between 6.8-7.2;
(6) solution send bacterial culture, requires aseptic;
(7) stored refrigerated;
(2) lycopene is added
(1) yolk, the 37 DEG C of water-baths of the compound cryoprotector of glycerine are thawed;
(2) lycopene takes out in refrigerator, gets lycopene 4.296 × 10 respectively
-4g-4.296 × 10
-3g incorporates in 0.01mL-0.1mL dimethyl sulfoxide (DMSO), makes the solution that content of lycopene is 80mmol/L,
(3) get the above-mentioned solution of 0.01mL-0.1mL to join in the yolk of 99.9mL-99.99mL, the compound cryoprotector of glycerine, wherein lycopene concentration is 8 μm of ol/L ~ 80 μm ol/L.
Adopt the Seminal plasma freezing method of the Seminal plasma cryoprotector containing lycopene, comprise the following steps:
(1) 7 DEG C of water-bath is thawed containing the Seminal plasma cryoprotector of lycopene;
(2) the Seminal plasma cryoprotector containing lycopene getting 1 part of volume joins in 3 parts of volume seminal fluid, dropwise adds Spin tubes simultaneously;
(3) after adding cryoprotector, mixed liquor is divided in sperm freezing pipe, and use Thermo7451 Programmed freezing instrument freezing, setting freezing procedure is as follows:
(1) 20 DEG C balances 5 minutes;
(2)-2 DEG C are down to from 20 DEG C, freezing rate-1 DEG C/min;
(3)-30 DEG C are down to from-2 DEG C, freezing rate-7.2 DEG C/min;
(4)-90 DEG C are down to from-30 DEG C, freezing rate-30 DEG C/min.
Tool of the present invention has the following advantages: lycopene is lipid fractions matter, is added directly in water-soluble protectant, can not form the protectant solution of stable homogeneous.Therefore, we select to dissolve lycopene and the organic solvent that can dissolve each other with water-soluble protectant as intermediate medium, enable the water-soluble dissolubility protectant of lycopene, reach better protective effect.Reduce in sperm frozen-thaw process and produce the damage of a large amount of active oxygen to sperm, therefore add polyphenoils in frozen solution: lycopene, and adopt a kind of intermediate medium better to the damage of antiactive oxygen to sperm, maintain normal morphology and the function of sperm.At present, report is not also had to take this kind of measure to be used in Seminal plasma cryoprotector by lycopene.
In semen freezing protection liquid, add certain density lycopene can reduce Mitochondrial oxidative damage, alleviate ROS and the oxidative stress of plasmalemmae of sperms is damaged, thus improve the anti-apoptotic ability of sperm.Improve anabiosis rate, motility rate, progressive sperm number and the normal morphology rate of frozen semen.
The present invention adds a certain amount of lycopene; human spermatogoa refrigerating effect is had clear improvement; human spermatogoa function assessment parameter after recovery: forward direction activity ratio, survival rate are significantly higher than control group (not adding lycopene); and normal morphology rate, DNA percentage of head rice and mitochondrial membrane potential are also apparently higher than control group; and sperm apoptosis rate is significantly lower than control group, thus prompting has more good safety to human spermatogoa quality protection.
Accompanying drawing explanation
Figure 1A is the result figure of the FCM detection Sperm Apoptosis of control group sample, Ly 0:43.26%;
Figure 1B is the result figure of the FCM detection Sperm Apoptosis of Ly5 μm of o/L group sample, Ly 5:35.68%;
Fig. 2 A is sperm mitochondrial membrane potential level view under sperm morphology and fluorescence microscope (X400) under the light microscopic of control group sample;
Fig. 2 B is sperm mitochondrial membrane potential level view under sperm morphology and fluorescence microscope (X400) under the light microscopic of Ly5 μm of o/L sample;
Fig. 3 is the protectant Cryopreservation Sperm progressive motility rate figure adding variable concentrations lycopene;
Fig. 4 is the protectant Cryopreservation sperm anabiosis rate figure adding variable concentrations lycopene;
Fig. 5 is the protectant Cryopreservation sperm normal morphology rate figure adding variable concentrations lycopene;
Fig. 6 is the protectant Cryopreservation sperm DNA percentage of head rice figure adding variable concentrations lycopene;
Fig. 7 is the protectant Cryopreservation sperm apoptosis rate figure adding variable concentrations lycopene.
Embodiment
Lycopene is preparing the application in Seminal plasma cryoprotector.
Lycopene is preparing the application that can improve in the Seminal plasma cryoprotector of Cryopreservation Sperm progressive motility rate.
Lycopene is preparing the application that can improve in the Seminal plasma cryoprotector of Cryopreservation sperm anabiosis rate.
Lycopene is preparing the application that can improve in the Seminal plasma cryoprotector of Cryopreservation sperm normal morphology rate.
Lycopene is preparing the application that can improve in the Seminal plasma cryoprotector of Cryopreservation sperm DNA percentage of head rice.
Lycopene is preparing the application that can reduce in the Seminal plasma cryoprotector of Cryopreservation sperm apoptosis rate.
Embodiment 1
Following material is contained containing in every 100mL Seminal plasma cryoprotector of lycopene:
Glucose 1.5g, trisodium citrate two water 1.3g, glycerine 20ml, lycopene 4.296 × 10
-4g, yolk 20ml, dimethyl sulfoxide (DMSO) 0.01mL, surplus is distilled water;
The preparation method of the Seminal plasma cryoprotector of described lycopene comprises the following steps:
(1) preparation of 99.99mL yolk, the compound cryoprotector of glycerine
(1) weigh 1.5g glucose and 1.3g trisodium citrate two water, add distilled water to 59.99mL;
(2) mix completely after adding 20mL glycerine, filter with 0.45 μm of millipore filter after dissolving completely;
(3) add the fresh yolk of 20mL, make suspension; Fresh yolk preparation: cleaning egg, shells, punctures egg membrane, draw yolk with syringe;
(4) to be put by made suspension in 56 DEG C of water-baths 30 minutes, concussion is stirred;
(5) detect the pH value of solution, make pH value 6.8;
(6) solution send bacterial culture, requires aseptic;
(7) stored refrigerated;
(2) add lycopene, lycopene available from Sigma, purity is greater than 99%:
(1) yolk, the 37 DEG C of water-baths of the compound cryoprotector of glycerine are thawed;
(2) lycopene takes out in refrigerator, gets lycopene 4.296 × 10 respectively
-4g incorporates in 0.01mL dimethyl sulfoxide (DMSO), makes the solution that content of lycopene is 80mmol/L,
(3) get the above-mentioned solution of 0.01mL to join in the yolk of 99.99mL, the compound cryoprotector of glycerine, wherein lycopene concentration is 8 μm of ol/L.
Adopt the Seminal plasma freezing method of the Seminal plasma cryoprotector containing lycopene, comprise the following steps:
(1) 7 DEG C of water-bath is thawed containing the Seminal plasma cryoprotector of lycopene;
(2) the Seminal plasma cryoprotector containing lycopene getting 1 part of volume joins in 3 parts of volume seminal fluid, dropwise adds Spin tubes simultaneously;
(3) after adding cryoprotector, mixed liquor is divided in sperm freezing pipe, and use Thermo7451 Programmed freezing instrument freezing, setting freezing procedure is as follows:
(1) 20 DEG C balances 5 minutes;
(2)-2 DEG C are down to from 20 DEG C, freezing rate-1 DEG C/min;
(3)-30 DEG C are down to from-2 DEG C, freezing rate-7.2 DEG C/min;
(4)-90 DEG C are down to from-30 DEG C, freezing rate-30 DEG C/min.
Embodiment 2
Following material is contained containing in every 100mL Seminal plasma cryoprotector of lycopene:
Glucose 1.5g, trisodium citrate two water 1.3g, glycerine 20ml, lycopene 1.074 × 10
-3g, yolk 20ml, dimethyl sulfoxide (DMSO) 0.025mL, surplus is distilled water;
The preparation method of the Seminal plasma cryoprotector of described lycopene comprises the following steps:
(1) preparation of 99.975mL yolk, the compound cryoprotector of glycerine
(1) weigh 1.5g glucose and 1.3g trisodium citrate two water, add distilled water to 59.975mL;
(2) mix completely after adding 20mL glycerine, filter with 0.45 μm of millipore filter after dissolving completely;
(3) add the fresh yolk of 20mL, make suspension; Fresh yolk preparation: cleaning egg, shells, punctures egg membrane, draw yolk with syringe;
(4) to be put by made suspension in 56 DEG C of water-baths 30 minutes, concussion is stirred;
(5) detect the pH value of solution, make pH value 6.8;
(6) solution send bacterial culture, requires aseptic;
(7) stored refrigerated;
(2) add lycopene, lycopene available from Sigma, purity is greater than 99%:
(1) yolk, the 37 DEG C of water-baths of the compound cryoprotector of glycerine are thawed;
(2) lycopene takes out in refrigerator, gets lycopene 1.074 × 10 respectively
-3g incorporates in 0.025mL dimethyl sulfoxide (DMSO), makes the solution that content of lycopene is 80mmol/L,
(3) get the above-mentioned solution of 0.025mL to join in the yolk of 99.975mL, the compound cryoprotector of glycerine, wherein lycopene concentration is 20 μm of ol/L.
Adopt the Seminal plasma freezing method of the Seminal plasma cryoprotector containing lycopene, comprise the following steps:
(1) 7 DEG C of water-bath is thawed containing the Seminal plasma cryoprotector of lycopene;
(2) the Seminal plasma cryoprotector containing lycopene getting 1 part of volume joins in 3 parts of volume seminal fluid, dropwise adds Spin tubes simultaneously;
(3) after adding cryoprotector, mixed liquor is divided in sperm freezing pipe, and use Thermo7451 Programmed freezing instrument freezing, setting freezing procedure is as follows:
(1) 20 DEG C balances 5 minutes;
(2)-2 DEG C are down to from 20 DEG C, freezing rate-1 DEG C/min;
(3)-30 DEG C are down to from-2 DEG C, freezing rate-7.2 DEG C/min;
(4)-90 DEG C are down to from-30 DEG C, freezing rate-30 DEG C/min.
Embodiment 3
Following material is contained containing in every 100mL Seminal plasma cryoprotector of lycopene:
Glucose 1.5g, trisodium citrate two water 1.3g, glycerine 20ml, lycopene 2.148 × 10
-3g, yolk 20ml, dimethyl sulfoxide (DMSO) 0.05mL, surplus is distilled water;
The preparation method of the Seminal plasma cryoprotector of described lycopene comprises the following steps:
(1) preparation of 99.95mL yolk, the compound cryoprotector of glycerine
(1) weigh 1.5g glucose and 1.3g trisodium citrate two water, add distilled water to 59.95mL;
(2) mix completely after adding 20mL glycerine, filter with 0.45 μm of millipore filter after dissolving completely;
(3) add the fresh yolk of 20mL, make suspension; Fresh yolk preparation: cleaning egg, shells, punctures egg membrane, draw yolk with syringe;
(4) to be put by made suspension in 56 DEG C of water-baths 30 minutes, concussion is stirred;
(5) detect the pH value of solution, make pH value 6.8;
(6) solution send bacterial culture, requires aseptic;
(7) stored refrigerated;
(2) add lycopene, lycopene available from Sigma, purity is greater than 99%:
(1) yolk, the 37 DEG C of water-baths of the compound cryoprotector of glycerine are thawed;
(2) lycopene takes out in refrigerator, gets lycopene 2.148 × 10 respectively
-3g incorporates in 0.05mL dimethyl sulfoxide (DMSO), makes the solution that content of lycopene is 80mmol/L,
(3) get the above-mentioned solution of 0.05mL to join in the yolk of 99.95mL, the compound cryoprotector of glycerine, wherein lycopene concentration is 40 μm of ol/L.
Adopt the Seminal plasma freezing method of the Seminal plasma cryoprotector containing lycopene, comprise the following steps:
(1) 7 DEG C of water-bath is thawed containing the Seminal plasma cryoprotector of lycopene;
(2) the Seminal plasma cryoprotector containing lycopene getting 1 part of volume joins in 3 parts of volume seminal fluid, dropwise adds Spin tubes simultaneously;
(3) after adding cryoprotector, mixed liquor is divided in sperm freezing pipe, and use Thermo7451 Programmed freezing instrument freezing, setting freezing procedure is as follows:
(1) 20 DEG C balances 5 minutes;
(2)-2 DEG C are down to from 20 DEG C, freezing rate-1 DEG C/min;
(3)-30 DEG C are down to from-2 DEG C, freezing rate-7.2 DEG C/min;
(4)-90 DEG C are down to from-30 DEG C, freezing rate-30 DEG C/min.
Embodiment 4
Following material is contained containing in every 100mL Seminal plasma cryoprotector of lycopene:
Glucose 1.5g, trisodium citrate two water 1.3g, glycerine 20ml, lycopene 4.296 × 10
-3g, yolk 20ml, dimethyl sulfoxide (DMSO) 0.1mL, surplus is distilled water;
The preparation method of the Seminal plasma cryoprotector of described lycopene comprises the following steps:
(1) preparation of 99.9mL yolk, the compound cryoprotector of glycerine
(1) weigh 1.5g glucose and 1.3g trisodium citrate two water, add distilled water to 59.9mL;
(2) mix completely after adding 20mL glycerine, filter with 0.45 μm of millipore filter after dissolving completely;
(3) add the fresh yolk of 20mL, make suspension; Fresh yolk preparation: cleaning egg, shells, punctures egg membrane, draw yolk with syringe;
(4) to be put by made suspension in 56 DEG C of water-baths 30 minutes, concussion is stirred;
(5) detect the pH value of solution, make pH value 6.8;
(6) solution send bacterial culture, requires aseptic;
(7) stored refrigerated;
(2) add lycopene, lycopene available from Sigma, purity is greater than 99%:
(1) yolk, the 37 DEG C of water-baths of the compound cryoprotector of glycerine are thawed;
(2) lycopene takes out in refrigerator, gets lycopene 4.296 × 10 respectively
-3g incorporates in 0.1mL dimethyl sulfoxide (DMSO), makes the solution that content of lycopene is 80mmol/L,
(3) get the above-mentioned solution of 0.1mL to join in the yolk of 99.9mL, the compound cryoprotector of glycerine, wherein lycopene concentration is 80 μm of ol/L.
Adopt the Seminal plasma freezing method of the Seminal plasma cryoprotector containing lycopene, comprise the following steps:
(1) 7 DEG C of water-bath is thawed containing the Seminal plasma cryoprotector of lycopene;
(2) the Seminal plasma cryoprotector containing lycopene getting 1 part of volume joins in 3 parts of volume seminal fluid, dropwise adds Spin tubes simultaneously;
(3) after adding cryoprotector, mixed liquor is divided in sperm freezing pipe, and use Thermo7451 Programmed freezing instrument freezing, setting freezing procedure is as follows:
(1) 20 DEG C balances 5 minutes;
(2)-2 DEG C are down to from 20 DEG C, freezing rate-1 DEG C/min;
(3)-30 DEG C are down to from-2 DEG C, freezing rate-7.2 DEG C/min;
(4)-90 DEG C are down to from-30 DEG C, freezing rate-30 DEG C/min.
Most important and the most frequently used index evaluating sperm freezing level is exactly the Cryopreservation rate of vigor after sperm freeze thawing is recovered and sperm; the sperm percentage most with propulsion ability is representative; therefore; the present invention designs and implements experimental study; lycopene is added in sperm freezing protecting agent formula conventional at present; form the formula of multiple different ratio, be applied to human spermatogoa freezing, whether the vigor after observation sperm freeze thawing recovery and function are improved.The not only progressive sperm percentage of the freezing front and back of observation and comparison, also observation and comparison freezing front and back sperm normal morphology rate, survival rate, sperm apoptosis rate and sperm mitochondrial membrane potential, these indexs can evaluate sperm function and sperm physiology and toxicological security preferably.
The present invention is the refrigerating effect of the sperm freezing protecting agent studying different lycopene concentration proportioning, explores through preliminary experiment, determines to adopt randomized complete-block design in conjunction with Repeated Measurements.
Select contribute the semen sample of smart person from No.1 Hospital of Jilin Univ.'s Jilin Province's human sperm bank year March in September, 2013 to 2014, amount to 25 parts, every part of seminal fluid is quadruplicate, mix after adding 1 frozen solution in 3 parts of seminal fluid, control group (Ly0) is not set to containing lycopene person, and Ly2, Ly5, Ly10, containing final concentration respectively in Ly20 experimental group mixed liquor is 2 μm of ol/L, 5 μm of ol/L, 10 μm of ol/L, the lycopene of 20 μm of ol/L, (use the protectant of the different lycopene concentration of embodiment 1-4 respectively: lycopene concentration is respectively: embodiment 1:8 μm of ol/L, embodiment 2:20 μm of ol/L, embodiment 3:40 μm of ol/L, embodiment 4:80 μm of ol/L), Cryopreservation semen routine analysis, the apoptosis rate of each group seminal fluid after adopting flow cytometry freeze thawing, each group Sperm progressive motility rate after adopting U.S. HAMILTON THORNE computer-assisted sperm analysis system to analyze freeze thawing, respectively sperm motility rate is organized after adopting nigrosine decoration method to analyze freeze thawing, each group sperm normal morphology rate after adopting improvement Papanicolaou's vaginal smear technique to analyze freeze thawing, utilize JC-1 tracer method detection line mitochondrial membrane potential.
Concrete process of the test is as follows:
After seminal fluid adds the protectant of different lycopene concentration; in freeze thawing seminal fluid, lycopene final concentration is respectively Ly0 μm o/L, Ly2 μm o/L, Ly5 μm o/L, Ly10 μm o/L, Ly20 μm of o/L; detect respectively freezing before and recovery after human spermatogoa following functions mathematic(al) parameter: forward direction activity ratio, survival rate, normal morphology rate, sperm apoptosis rate and mitochondrial membrane potential, calculate anabiosis rate.
(1) detection of forward direction activity ratio: adopt the U.S. to produce full-automatic computer aided pass design and detect human spermatogoa concentration and vigor, the results are shown in accompanying drawing 3, table 1.
Sperm motility before and after table 1 semen freezing recovery and PR compare (x ± s, n=25)
(2) detection of survival rate: get semen sample 50 μ l after freeze thawing, mix with isopyknic Yihong-nigrosine suspension, wait for 30 seconds;
Before repeated sampling, again mix semen sample, sampling mixes with Yihong-aniline black liquor.Use each suspension, smear made by slide, air drying; Check immediately after drying, bright-field microscope checks under × 1000 times of oily mirrors often opens slide; By laboratory count device, counting staining sperm cells (Necrospermia) and non-staining sperm (sperm of living); Each repeated sample assesses 200 sperms, to reach acceptable low sampling error, calculates percentage.The results are shown in accompanying drawing 4.
(3) detection of sperm morphology mathematic(al) parameter: according to the method smear of WHO the 5th edition, then dye according to improvement pap staining program, fluorescence microscope, calculates normal morphology rate, the results are shown in accompanying drawing 5.
(4) detection of DNA percentage of head rice: according to TUNEL kit step, semen sample after freeze thawing takes a morsel sample (10 μ l) routine smear, carry out TUNEL dyeing, observe sperm DNA integrity, its principle is: the indentation, there that DNA chain can produce in fracture due to damage exposes 3 '-OH end, sweet acid (dUTP) is urinated under the effect of DNA end transferase (TdT enzyme) with fluorescein-labeled deoxidation, can react with 3 '-OH end, fluorescent effect is produced with the laser excitation of respective wavelength, if normal cell is due to DNA chain not damaged, then dyeing is shown as blue-fluorescence, if DNA chain damages, then shown in green fluorescence.TUNEL cell positive ratio is the percentage that apoptosis spermoblast accounts for total sperm sum.The results are shown in accompanying drawing 6.
(5) mensuration of sperm apoptosis rate after freeze thawing: recovered by frozen semen sample, takes out sample 400 μ l and adds PBS centrifuge washing 2 times, remove refining and supernatant, make 1 × 10 with incubation buffer 1ml resuspension
5the sperm suspension of/ml, gets 100 μ l cell suspensions and puts into test tube, and add FITC-Annexin V 10 μ l and PI 5 μ l and dye, under room temperature, lucifuge adds 400 μ l incubation buffer after leaving standstill 15min, 1h in-flow cell instrument FCM measurement result.The fluorescence signal of all mark sperms all passes through flow cytomery.Each detection at least 10000 sperms.Forward scattering/lateral scattering is adopted to establish door to get rid of the interference of cell fragment and cell aggregation.First by scattered light signal sorting sperms and cell fragment, and then distinguish living cells, dead cell and apoptotic cell on bivariate fluorescence signal scatter diagram.Excitation wavelength is 488nm, and green fluorescence (480 ~ 530nm) adopts FL1 Air conduct measurement, and red fluorescence (580 ~ 630nm) adopts FL2 Air conduct measurement, and positive cell ratio peace all fluorescence intensity adopts CellQuest software to analyze.Dyeed by FITC-Annexin V/PI, sperm colony can be divided into 4 subgroups: that left lower quadrant shows is AnnexinV
-/ PI
-sperm, namely without the sperm alive that phosphatidylserine (PS) is aobvious outward; That right lower quadrant shows is AnnexinV
+/ PI
-sperm, the sperm alive of the early apoptosis namely having PS to show outward; That right upper quadrant shows is AnnexinV
+/ PI
+sperm, namely has the late apoptic sperm that PS shows outward; That left upper quadrant shows is AnnexinV
-/ PI
+sperm, namely without the Necrospermia that PS shows outward, accompanying drawing 1A and Figure 1B.
(6) get 400 μ l in sample after mitochondrial membrane potential detects Cryopreservation, the centrifugal 5min of 3000r/min removes supernatant, adds JC-1500 μ l solution and shakes up, at 37 DEG C, 5%CO
2incubator incubation 10min, the centrifugal 5min of 3000r/min removes supernatant, wash 2 times by 1 × analysis buffer and remove excess dyestuff, finally use the resuspended precipitation of PBS, get wherein 10 μ l sample drop push jack on slide, staining of sperm situation is visually observed with fluorescence microscope, this experiment adopts JC-1 fluorescence probe method to detect the mitochondrial membrane potential of sperm, when mitochondrial membrane potential is higher, JC-1 is gathered in mitochondrial matrix, form polymer and produce red fluorescence, when mitochondrial membrane potential is lower, JC-1 can not be gathered in mitochondrial matrix, now JC-1 is monomer, produce green fluorescence.Normal cell membrane potential is higher, and Microscopic observation is red; When there is apoptosis, film potential declines, and red fluorescence dies down, until Microscopic observation is less than fluorescence, accompanying drawing 2A and Fig. 2 B, left side is illustrated as sperm morphology under light microscopic, and right diagram is sperm mitochondrial membrane potential level under fluorescence microscope.Every part of sample at least continuous counter 200 sperms, positive number of sperm is divided by number sum ratio calculated that is negative in the same visual field and positive sperm.
Data results in Fig. 7 shows: embodiment 2,3, after 4 Cryopreservations, sperm viability is apparently higher than embodiment 1 and comparative examples (P<0.05), and sperm morphological index and sperm DNA percentage of head rice are obviously better than other formula (P<0.05).So the lycopene of finite concentration scope can improve the effect of sperm freezing protecting agent.Especially, when lycopene concentration is 5 μm of ol/L, after Cryopreservation, sperm viability is obviously higher.
To sum up after freeze thawing, each group sperm motility parameters comparatively fresh seminal parameters all obviously declines (P < 0.01), add lycopene group, sperm freezing anabiosis rate, motility rate, progressive sperm number and normal morphology rate are all significantly higher than control group (P < 0.05), wherein more outstanding with Ly5 group effect; Sperm apoptosis rate [(25.68 ± 4.36) %] comparatively control group [(33.26 ± 4.78) %] significantly decline (P < 0.05) after the freeze thawing of Ly5 group; Ly5 group mitochondrial membrane potential level [(66.18 ± 14.23) %] is significantly higher than control group [(55.24 ± 12.31)], P<0.05.
Claims (9)
1. lycopene is preparing the application in Seminal plasma cryoprotector.
2. lycopene is preparing the application that can improve in the Seminal plasma cryoprotector of Cryopreservation Sperm progressive motility rate.
3. lycopene is preparing the application that can improve in the Seminal plasma cryoprotector of Cryopreservation sperm anabiosis rate.
4. lycopene is preparing the application that can improve in the Seminal plasma cryoprotector of Cryopreservation sperm normal morphology rate.
5. lycopene is preparing the application that can improve in the Seminal plasma cryoprotector of Cryopreservation sperm DNA percentage of head rice.
6. lycopene is preparing the application that can reduce in the Seminal plasma cryoprotector of Cryopreservation sperm apoptosis rate.
7., containing the Seminal plasma cryoprotector of lycopene, it is characterized in that every 100mL contains following material:
Glucose 1.5g, trisodium citrate two water 1.3g, glycerine 20ml, lycopene 4.296 × 10
? 4g ?4.296 × 10
? 3g, yolk 20ml, dimethyl sulfoxide (DMSO) 0.01mL ?0.1mL, surplus is distilled water.
8., as claimed in claim 7 containing the preparation method of the Seminal plasma cryoprotector of lycopene, comprise the following steps:
(1) 99.9mL ?99.99mL yolk, the compound cryoprotector of glycerine preparation
(1) weigh 1.5g glucose and 1.3g trisodium citrate two water, add distilled water to 59.9mL ?59.99mL;
(2) mix completely after adding 20mL glycerine, filter with 0.45 μm of millipore filter after dissolving completely;
(3) add the fresh yolk of 20mL, make suspension;
(4) to be put by made suspension in 56 DEG C of water-baths 30 minutes, concussion is stirred;
(5) detect the pH value of solution, make pH value 6.8 ?between 7.2;
(6) solution send bacterial culture, requires aseptic;
(7) stored refrigerated;
(2) lycopene is added
(1) yolk, the 37 DEG C of water-baths of the compound cryoprotector of glycerine are thawed;
(2) lycopene takes out in refrigerator, gets lycopene 4.296 × 10 respectively
? 4g ?4.296 × 10
? 3g incorporate 0.01mL ?in 0.1mL dimethyl sulfoxide (DMSO), make the solution that content of lycopene is 80mmol/L,
(3) get 0.01mL ?the above-mentioned solution of 0.1mL join 99.9mL ?the yolk of 99.99mL, in the compound cryoprotector of glycerine, wherein lycopene concentration is 8 μm of ol/L ~ 80 μm ol/L.
9. adopt as claimed in claim 7 containing the Seminal plasma freezing method of the Seminal plasma cryoprotector of lycopene, comprise the following steps:
(1) 7 DEG C of water-bath is thawed containing the Seminal plasma cryoprotector of lycopene;
(2) the Seminal plasma cryoprotector containing lycopene getting 1 part of volume joins in 3 parts of volume seminal fluid, dropwise adds Spin tubes simultaneously;
(3) after adding cryoprotector, mixed liquor is divided in sperm freezing pipe, and use Thermo7451 Programmed freezing instrument freezing, setting freezing procedure is as follows:
(1) 20 DEG C balances 5 minutes;
(2)-2 DEG C are down to from 20 DEG C, freezing rate-1 DEG C/min;
(3)-30 DEG C are down to from-2 DEG C, freezing rate-7.2 DEG C/min;
(4)-90 DEG C are down to from-30 DEG C, freezing rate-30 DEG C/min.
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| CN114223649A (en) * | 2021-12-24 | 2022-03-25 | 暨南大学 | Application of anthocyanin in sperm cryopreservation and sperm cryopreservation liquid |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106508886A (en) * | 2016-09-14 | 2017-03-22 | 西北农林科技大学 | Application of lycopene and alpha-lipoic acid as mammal sperm cryoprotectant |
| CN106508886B (en) * | 2016-09-14 | 2019-11-12 | 西北农林科技大学 | Lycopene and application of the alpha-lipoic acid as mammal semen cryopreservation agent |
| CN106538510A (en) * | 2016-09-22 | 2017-03-29 | 西北农林科技大学 | The application of lycopene and sesamol compatibility in domestic animal semen cryopreservation agent |
| CN114223649A (en) * | 2021-12-24 | 2022-03-25 | 暨南大学 | Application of anthocyanin in sperm cryopreservation and sperm cryopreservation liquid |
| CN114885940A (en) * | 2022-06-01 | 2022-08-12 | 重庆医科大学附属第一医院 | Application of Fer-1 in preparation of semen cryoprotectant |
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