CN102450248A - Sperm freezing method and freezing liquid thereof - Google Patents
Sperm freezing method and freezing liquid thereof Download PDFInfo
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- CN102450248A CN102450248A CN2010105195667A CN201010519566A CN102450248A CN 102450248 A CN102450248 A CN 102450248A CN 2010105195667 A CN2010105195667 A CN 2010105195667A CN 201010519566 A CN201010519566 A CN 201010519566A CN 102450248 A CN102450248 A CN 102450248A
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Abstract
The invention relates to a sperm freezing method which mainly comprises the following steps: 1) diluting and well mixing sperm and freezing liquid according to a ratio of 1:1; 2) slowly cooling at 4 DEG C, after 3 hours, individually adding the mixture in plastic tubules of 0.25-0.35 ml and sealing, putting on a suspended object with a distance from a liquid nitrogen surface of 4-6 cm at a temperature of from -90 to -95 DEG C; 3) putting the tubules into the liquid nitrogen after 10-15 minutes. The sperm freezing method provided by the invention has simple operations, and the frozen-thawed sperm still has high fertilization ability and an high embryonic development rate.
Description
Technical field
The invention belongs to bioengineering field, relate to a kind of freezing processing method, relate in particular to a kind of freezing method and freezing liquid thereof of sperm.
Background technology
Intracytoplasmic sperm injection technology (ICSI) is as a kind of means of assisted fertilization in the egg mother cell kytoplasm; In short decades, obtained swift and violent development; Character and scope with regard to the ICSI application; The application of current I CSI can be classified as two big types, and the one, it can solve a series of sterile problems that reason caused such as factor of sperm own and salpingemphraxis, so be widely used in the sterility treatment of reproductive medicine; The 2nd, be used for bioengineering field as a kind of biotechnology, the research of Developmental Biology basic theories such as herding production, the conservation of wildlife and mechanism of fertilization is all had very important significance.
The ICSI technology generally includes preparation or preliminary treatment, the interior microinjection of sperm kytoplasm, the activation of injection ovum and the observation and the evaluation of the outer developmental state of injection oophyte of sperm and ovum.And the quality of sperm is the successful key factor of ICSI technology.
In the last few years, although people are deep day by day to the research of sperm freezing, the survival of frozen sperm and fertilization did not have significant improvement as yet.
Summary of the invention
The object of the present invention is to provide a kind of method of frozen sperm, it mainly comprises step:
1) seminal fluid and freezing liquid are pressed 1: 1 dilution proportion mixing;
2) 4 ℃ of slowly coolings with 0.25～0.35ml plastic straw coating-dividing sealing, place on the suspension behind the 3h, and apart from liquid nitrogen surface 4～6cm, its temperature is-90～-95 ℃;
3) drop in the liquid nitrogen after 10～15 minutes.
Freezing liquid described in the step 1) contains: deionized water, TrisHCL, citric acid, glucose, yolk, glycerine.Described freezing liquid obtains through following method: add 3.52gTrisHCL, 1.96g citric acid, 1.59g glucose mixing in the 100ml deionized water as basal liquid; Adding 20ml yolk, 4ml glycerin obtained get the 100ml freezing liquid in the 76ml basal liquid.
The present invention also provides a kind of freezing liquid, and it mainly contains: deionized water, TrisHCL, citric acid, glucose, yolk, glycerine.Comprise basal liquid and 20ml yolk and 4ml glycerine that 76ml is made up of deionized water, TrisHCL, citric acid and glucose in every 100ml freezing liquid; The ratio of each component is deionized water: TrisHCL in the said basal liquid: citric acid: glucose=100ml: 3.52g: 1.96g: 1.59g.Described freezing liquid prepares through following method: add 3.52gTrisHCL, 1.96g citric acid, 1.59g glucose mixing in the 100ml deionized water as basal liquid; Adding 20ml yolk, 4ml glycerin obtained get the 100ml freezing liquid in the 76ml basal liquid.
Described freezing liquid can be used for frozen sperm.
Sperm freezing method provided by the invention, simple to operate, and also the sperm after the freeze thawing still has higher fertility and embryo development rate.
For let above and other objects of the present invention, feature and advantage can be more obviously understandable, hereinafter is special lifts preferred embodiment, and conjunction with figs., elaborates as follows.
Description of drawings
Fig. 1 is the operation board sketch map;
Fig. 2 is the female-male pronucleus and the first diode sketch map;
Fig. 3 is a 2-cell stage sketch map;
Fig. 4 is a 4-cell stage sketch map;
Fig. 5 mulberry body sketch map;
Fig. 6 is the blastaea sketch map;
Fig. 7 is an expansion blastaea sketch map;
Fig. 8 is the hatched blastocyst sketch map;
Fig. 9 is embryo's sketch map of living after the freeze thawing;
Figure 10 is embryo dead after the freeze thawing;
Figure 11 is the micromanipulation sketch map;
Figure 12 is the young rabbit of birth.
Embodiment
Embodiment 1The preparation of sperm
1. fresh spermatozoa:
(Shanghai Slac Experimental Animal Co., Ltd. provides, credit number: SCXK for 6～8 monthly age new zealand rabbits, SPF level to get rabbit<hu>2004-0005) fresh semen adds D-PBS liquid three times then slowly with D-PBS liquid (being purchased) 2000r/min centrifuge washing, is placed on 38.5 ℃, 5%CO
2, the slow upper reaches 10 minutes in the incubator under 100% humidity.
2. the sperm of freeze thawing:
(prescription of freezing liquid is: add 3.52gTrisHCL, 1.96g citric acid, 1.59g glucose mixing in the 100ml deionized water as basal liquid to gather rabbit seminal fluid and freezing liquid; The 100ml freezing liquid: add 20ml yolk, 4ml glycerine in the 76ml basal liquid, the mixing packing, 4 ℃ of refrigerators are preserved.) 1: 1 dilution mixing, 4 ℃ of slowly coolings with 0.25～0.30ml plastic straw coating-dividing sealing, place (apart from liquid nitrogen surface 4cm ,-90--95 ℃) on the levitron behind the 3h, drop into liquid nitrogen after 10 minutes.The tubule that the sperm that will thaw is housed is placed 42 ℃ of water-baths 20 seconds.Rabbit freeze thawing seminal fluid is directly put into D-PBS liquid, at 38.5 ℃, 5%CO
2, the slow upper reaches 10 minutes in the incubator under 100% humidity.
Embodiment 2The preparation of egg mother cell
1. fresh egg mother cell
The female rabbit of donor is the primiparity new zealand rabbit about 6-8 monthly age, body weight 3kg, presses the method for Kennelly and Foote (1965), i.e. 2 (12h at interval) each hypodermic injection FSH50IU/ (production of the Ningbo second hormone factory) every day for three days on end; 12h behind the last injection FSH; Auricular vein HCG injection (production of the Ningbo second hormone factory) 100IU/ only and with the mating of ligation buck, HCG injection back 14,16,18h get ovum, under aseptic condition, cut oviduct; That draws 10ml dashes ovum liquid; By horn mouth to palace pipe jointing portion direction towards ovum, every side, is placed on ovarian cumulus egg mother cell complex in the Eppendorf pipe that the D-PBS liquid that contains 0.2% hyaluronidase is housed towards ovum liquid with 5mL; Concussion digestion 5min removes granular cell on turbine mixer; Under stereomicroscope, pick egg mother cell,, put into 38.5 ℃, 5%CO then with culture fluid washing three times through enzymic digestion
2, under 100% humidity in the above culture fluid droplet of balance 12h, up to microinjection.
2. the egg mother cell of freeze thawing
1) egg mother cell is freezing
About 2h room temperature is transferred to 25 ℃ before freezing, simultaneously with all solution and test tool balance 1～2h under 25 ℃ of room temperatures.Adopt 0.25～0.30ml plastic straw (France), use the marking pen mark, be connected with syringe with thin emulsion tube then.Suck 0.5mol sucrose solution (5.5cm) → air (1.5cm) → vitrification solution (0.5cm) → air (0.5cm) → vitrification solution (1.5cm) in order; Be placed in parallel on the operating desk; Egg mother cell moved into wash in the vitrification solution once; Then 10 pieces of egg mother cells are placed the vitrification solution of tubule, egg mother cell sucks air (0.5cm) → vitrification solution (0.5cm) → air (0.5cm) after importing successively again, inhales 0.5mol sucrose solution (1cm) at last.With sealing machine plastic straw is sealed.Tubule one end that will contain egg mother cell directly drops into liquid nitrogen; Second half is stifling freezing with liquid nitrogen gas; Treat to change over to after sucrose partly freezes the medium-term and long-term preservation of liquid nitrogen container; Whole process is 1 minute till from egg mother cell immigration vitrification solution to tubule input liquid nitrogen, if room temperature transfers to 20 ℃, and then whole process need 2 minutes.
2) egg mother cell thaws
After plastic straw takes out from liquid nitrogen; Place 25 ℃ of water-baths to swing about 10 seconds gently fast, treat that the sucrose solution section becomes the taking-up of transparent back by milky in the tubule, inhale with blotting paper and remove the tubule surface moisture; Cut off sealing of tubule two ends; There is the syringe of 1mL0.5mol sucrose solution that the tubule content is poured in the surface plate with inhaling, at stereomicroscope recover egg mother cell, after the egg mother cell of recovery moves into 0.5mol sucrose solution inner equilibrium removal in 5 minutes ethylene glycol and ficoll; Change over to again in the D-PBS liquid, treat that then egg mother cell recovers to continue for 2-3 time to cultivate with the culture fluid washing again after the normal morphology.
Put into 38.5 ℃, 5%CO after thawing
2, 3h at least in the above culture fluid droplet of balance 12h under 100% humidity, operate then.
Embodiment 3With sperm injection in egg mother cell
1. the making of operation board
The design of operation board is by " the style design that mice embryonic operation experiments handbook third edition ICSI chapters and sections are introduced; Promptly cover and make 4-5 row operation droplet successively in the 90mm Tissue Culture Dish; Every row operates drop and is dripped to drip with three 5ulmTCM199 by three 3ul 10%PVP (polyvinyl pyrrolidone) and form, and draws upper strata sperm that 2ul swims and joins during second PVP drip, and ovum places the 4th mTCM199 to drip; Usefulness is dripped in the cleaning that all the other droplets are all done holding pipe and entry needle in the middle of the dish; After all operation is dripped and carried out, cover the whole operation dish with the 3-5ml paraffin oil at last, the design pattern that the operation board operation is dripped is seen figure (Fig. 1).
2.PVP the adding of sperm in dripping
Size according to sperm concentration during PVP (1gPVP pours into to dispose in the 10mlD-PBS liquid and forms) drips adds in suitable ratio, mixes the 2.0ul sperm suspensions during each PVP drips, and concrete operations are the sperm free liquid with 10ul liquid-transfering gun absorption 2.0ul; Under stereomicroscope, the rifle head is deep into during PVP drips, blow out during sperm suspensions drips to PVP; The pressure-vaccum operation for several times repeatedly; Sperm is mixed in PVP drips, after sperm adds, cover paraffin oil.
3. the dress pin and the debugging of micromanipulation system
When the operating system entry needle; Draw 1～2ul mercury with an elongated mercury plastic suction pipe of front end; The part elongated mercury suction pipe front end stretches into into entry needle from the rear end of entry needle, slowly blows out the mercury in the suction pipe in the position from entry needle latter end 1.0～1.5cm, and mercury length in needle tubing is got final product between 2～3mm; Mercury is connected to entry needle on the Piezo motion arm after irritating well.
Before the injection; Move into 5-10 piece of egg mother cell in the operation drop with picking up the ovum pin, grasp a sperm with entry needle then and put into the 3rd PVP drop, streak rapidly at sperm tail with needle point and brake; See that the afterbody discounting gets final product, and sucks in the entry needle from afterbody subsequently.First polar body is at 12 o ' clock positions during injection, and from the position inserting needle at 3 o'clock, it is 2 * 2 or 3 * 2 that intensity and frequency parameter are set, and excites 1 to 2 pulse breakdown oolemma; Continue inserting needle simultaneously with sperm careful be pushed into the needle point place, insert the egg mother cell depths until needle point, when almost pressing close to the offside plasma membrane; The shelves of transposing parameter to 1 * 1 are worn the egg mother cell plasma membrane with the impact of Piezo weak pulse, and sperm is spued; Resorption injects excess liquid in the ovum, and a shot is accomplished in the withdraw of the needle gently.Operation successively, per injection finishes and in 2min, accomplishes from beginning to brake injection, and all micromanipulations are at room temperature accomplished.
4. the criterion of fertilized egg and lonely female activation ovum
Injection back 6-8h inspection protokaryon and second polar body form situation, have only when 2 protokaryons, 2 polar bodys (2PN, 2PB) occurring, to be judged to be fertilization; When 1 protokaryon, 1 polar body (1PN, 1PB), 1 protokaryon, 2 polar bodys (1PN, 2PB), 2 protokaryons, 1 polar body (2PN, 1PB) occurring, be judged to be activation.
Embodiment 4The cultivation of fertilized egg
After the microinjection, the injection ovum moves in the good culture fluid droplet of balance after culture fluid washing three times, is placed on 38.5 ℃, 5%CO
2, cultivated 3-4 days in the incubator under 100% humidity, cultivate and observe protokaryon behind 6～8h and form situation with second polar body, whenever observe also record spilting of an egg situation at a distance from 12h, 24h changes liquid at interval, in good time photograph.
Embodiment 5Embryo cryopreservation with thaw
Method is with the freezing and defreezing method of the egg mother cell among the embodiment 2.
Embodiment 6Embryo transplantation
The fresh ICSI embryo or the embryo of freeze thawing; The TCM199 culture fluid (add 2mlFBS, 0.00275g Sodium Pyruvate, 0.00075gEDTA, 0.0015g penicillin, 0.001g streptomycin among the 18mlTCM199, abundant mixing, transferring pH is 7.3; 0.22um filter filters packing; 4 ℃ of refrigerators are preserved) in cultivated 2-3 hour, the normal embryo of form supplies to transplant usefulness, acceptor is false pregnancy 60h after the HCG injection, gave birth to the adult female rabbit about a tire, physical health, body weight 3.5kg.Recipient rabbits is fasting 12h before operation.At first with new (the i.e. 846 mixture) intramuscular anesthesia of 0.5mL speed dormancy; Treat Baoding behind the rabbit anesthesia, cropping respectively, sterilization, surgical openings near two the part between the ribs and the hips portions expose the uterus; On uterine body, prick an aperture with syringe needle; Ovum shifting tube with the head passivation moves into the embryo in the uterus then, and every side uterus moves into 7-8 piece of embryo, every female rabbit 15-16 piece embryo.The whole surgery process is carried out under aseptic condition, accomplishes operation within a short period of time (40-50min) as far as possible, to the meticulous feeding and management of the female rabbit of postoperative.
Embodiment 7Interpretation of result
Adopt the card side (x among the SSPS 13.0
2) check is carried out significance analysis to the result, is significance of difference criterion with P＜0.05.
Please with reference to Fig. 2～12, wherein Fig. 2 is the female-male pronucleus and the first diode sketch map; Fig. 3 is a 2-cell stage sketch map; Fig. 4 is a 4-cell stage sketch map; Fig. 5 mulberry body sketch map; Fig. 6 is the blastaea sketch map; Fig. 7 is an expansion blastaea sketch map; Fig. 8 is the hatched blastocyst sketch map; Fig. 9 is embryo's sketch map of living after the freeze thawing; Figure 10 is embryo dead after the freeze thawing; Figure 11 is the micromanipulation sketch map; Figure 12 is the young rabbit of birth.
1. different ovum is got ovum period in age:
HCG injection back 14,16,18h get ovum, inject with new fresh and alive sperm, activate the method with mechanical stimulus, and first polar body is at 12 o ' clock positions, and the position inserting needle at 3 o'clock divides three groups to carry out, and every group is repeated 3 times, add up its fertilization rate, blastaea rate.The result sees table 1.
Can find out that from table 1 hCG injection back 14h gets ovum, its spilting of an egg rate, mulberry body rate and blastaea rate (82.2%, 72.9% and 62.2%) be all than the height of 16h (75.9%, 70.0% and 53.3%), but difference not significantly (P＞0.05); Can not the spilting of an egg after the injection of ovum that 18h gets, with 14h and 16h significant difference (P＜0.05).It is thus clear that, after the HCG injection 14h and 16h get ovum can, but 14h gets ovum the best.
Table 1
Annotate: same letter difference is remarkable (p＞0.05) not, different alphabetical significant differences (p＜0.05)
2. ovum is injected in intracytoplasmic sperm injection and activation:
Match grouping according to injection operation situation and different activation processing methods, sperm is with new fresh and alive sperm, and ovum is used fresh egg mother cell, divides three groups, uses mechanical stimulus for first group; Second group is 5umol/L ionomycin (5min)+2mmol/L6-DMAP (3h); The 3rd group is false injection control group; The egg mother cell of injection back survival is assigned to each group at random; Each processed group injection back Activation of Oocyte effect weighs with Activation of Oocyte rate, fertilization rate and parthenogenetic development rate behind the ICSI that (egg mother cell forms a protokaryon at least or the spilting of an egg takes place and counts activation, forms female-male pronucleus and counts fertilization; Under the situation of no sperm injection, form two protokaryons at least or the spilting of an egg take place and count parthenogenetic development), every group is repeated 3 times.The result sees table 2.
Can find out that from table 2 mechanical stimulus group and ionomycin+6-DMAP organizes spilting of an egg rate (82.2% and 81.1%), mulberry body rate (72.9% and 66.2%) difference is remarkable (P＞0.05) not, but blastaea rate (51.3% and 62.3%) significant difference (P＜0.05).Ionomycin+6-DMAP group embryo morphology is better, and the blastaea rate is higher, and active mode ionomycin+6-DMAP is more suitable in the activation of rabbit ICSI ovum.
Table 2
Annotate: same letter difference is remarkable (p＞0.05) not, different alphabetical significant differences (p＜0.05) ,+sperm injection;-do not inject sperm
3. cultivate the ICSI rabbit embryonic with culture fluid:
Cultivate with TCM199+10%FBS+1.25mM Sodium Pyruvate+0.1mM EDTA culture fluid, repeat 3 times, add up its developmental rate.The result sees table 3.
From table 3, can find out 2-cell rate (84.5% and 79.0%) difference not remarkable ((P＞0.05), but mulberry body rate (69.0% and 58.0%) and blastaea rate (57.1% and 38.7%) significant difference (P＜0.05) in A group and the B group.
Table 3
Annotate: same letter difference is remarkable (p＞0.05) not, different alphabetical significant differences (p＜0.05)
4.ICSI the ectogenesis of back rabbit embryonic:
Ovum is with the fresh egg mother cell of rabbit, and control group is with the new fresh and alive sperm of rabbit, and experimental group is that the sperm alive after freezing carries out ICSI, experimentizing on table 1～3 results' basis, and every group is repeated 3 times, adds up its ectogenesis rate.The result sees table 4.
From table 4, can find out fresh spermatozoa group and freeze thawing live sperm group spilting of an egg rate (81.1% and 68.8%) and blastaea rate (62.3% and 40.4%) significant difference (P＜0.05), and mulberry body rate (66.2% and 61.9) difference not remarkable (P＞0.05).It is thus clear that the rabbit sperm after freezing still has extraordinary fertility.
Table 4
Annotate: same letter difference is remarkable (p＞0.05) not, different alphabetical significant differences (p＜0.05)
5. the fertilization situation of the rabbit oocyte of freeze thawing:
Sperm is with fresh rabbit sperm, and control group is fresh rabbit oocyte, and experimental group is that the egg mother cell of living in freezing back carries out ICSI, on table 1～3 results' basis, experimentizes, and every group is repeated 3 times, add up its ectogenesis rate.The result sees table 5.
Can find out that from table 5 the rabbit oocyte spilting of an egg poor ability after freezing can not continue to grow blastaea.
Table 5
Annotate: same letter difference is remarkable (p＞0.05) not, different alphabetical significant differences (p＜0.05)
Can find out that from The above results the rabbit sperm after the freeze thawing still has higher fertility and embryo development rate.
Though the present invention discloses as above with preferred embodiment; Right its is not in order to limit the present invention; Any person of ordinary skill in the field; In spirit that does not break away from the present invention and scope, when can doing a little change and improvement, so the present invention's protection domain is as the criterion when looking the claim person of defining.
Claims (7)
1. the freezing method of a sperm is characterized in that, comprises step:
1) seminal fluid and freezing liquid are pressed 1: 1 dilution proportion mixing;
2) 4 ℃ of slowly coolings with 0.25～0.35ml plastic straw coating-dividing sealing, place on the suspension behind the 3h, and apart from liquid nitrogen surface 4～6cm, its temperature is-90～-95 ℃;
3) drop in the liquid nitrogen after 10～15 minutes.
2. method according to claim 1 is characterized in that, the freezing liquid described in the step 1) contains: deionized water, TrisHCL, citric acid, glucose, yolk, glycerine.
3. method according to claim 2 is characterized in that, described freezing liquid obtains through following method: add 3.52gTrisHCL, 1.96g citric acid, 1.59g glucose mixing in the 100ml deionized water as basal liquid; Adding 20ml yolk, 4ml glycerin obtained get the 100ml freezing liquid in the 76ml basal liquid.
4. a freezing liquid is characterized in that, contains: deionized water, TrisHCL, citric acid, glucose, yolk, glycerine.
5. freezing liquid according to claim 4 is characterized in that, comprises basal liquid and 20ml yolk and 4ml glycerine that 76ml is made up of deionized water, TrisHCL, citric acid and glucose in every 100ml freezing liquid; The ratio of each component is deionized water: TrisHCL in the said basal liquid: citric acid: glucose=100ml: 3.52g: 1.96g: 1.59g.
6. the preparation method of the described freezing liquid of claim 5 is characterized in that, comprises step: add 3.52gTrisHCL, 1.96g citric acid, 1.59g glucose mixing in the 100ml deionized water as basal liquid; Adding 20ml yolk, 4ml glycerin obtained get the 100ml freezing liquid in the 76ml basal liquid.
7. claim 4 or 5 application of described freezing liquid in frozen sperm.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104705287A (en) * | 2015-01-30 | 2015-06-17 | 王宝燕 | Erythrocyte cryopreservation liquid and application thereof |
| CN113812396A (en) * | 2021-09-28 | 2021-12-21 | 广东海洋大学 | Duck essence cryopreservation diluent |
| CN114009427A (en) * | 2021-12-11 | 2022-02-08 | 江苏省农业科学院 | Rabbit semen cryopreservation diluent, preparation method, rabbit semen cryopreservation method and application |
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2010
- 2010-10-26 CN CN2010105195667A patent/CN102450248A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104705287A (en) * | 2015-01-30 | 2015-06-17 | 王宝燕 | Erythrocyte cryopreservation liquid and application thereof |
| CN113812396A (en) * | 2021-09-28 | 2021-12-21 | 广东海洋大学 | Duck essence cryopreservation diluent |
| CN114009427A (en) * | 2021-12-11 | 2022-02-08 | 江苏省农业科学院 | Rabbit semen cryopreservation diluent, preparation method, rabbit semen cryopreservation method and application |
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Application publication date: 20120516 |