CN110547290B - Vitrification freezing method for pig oocyte - Google Patents
Vitrification freezing method for pig oocyte Download PDFInfo
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- CN110547290B CN110547290B CN201910788513.6A CN201910788513A CN110547290B CN 110547290 B CN110547290 B CN 110547290B CN 201910788513 A CN201910788513 A CN 201910788513A CN 110547290 B CN110547290 B CN 110547290B
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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
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- 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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Abstract
The invention provides a vitrification freezing technology of porcine oocytes, which comprises the following steps: preparing a liquid nitrogen box for freezing porcine oocytes; preparing in vitro maturation liquid (L-IVM) of porcine oocytes and operating liquid; manufacturing a freezing operation panel; an oral suction tube for oocyte operation; cryotop for freezing porcine oocytes; selecting frozen oocytes; freezing and thawing pig oocyte Cryotop; and (5) judging after the pig oocytes are frozen. The vitrification freezing technology of the pig oocyte is simple and effective, the survival rate of the oocyte after freezing by the method is 40.09%, the aim of freezing preservation is achieved, the damage to the oocyte caused by temperature reduction is avoided, the obstacle of cross-regional germplasm resource communication can be solved, and the local variety pig is protected.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a vitrification freezing method for porcine oocytes, which is used for more efficiently performing vitrification freezing on the porcine oocytes so as to achieve the aim of preservation.
Background
The preservation of mammalian oocytes has occurred as early as 1958, until after 20 years, a low temperature biological study was performed on the oocytes of mice, and after freezing and thawing, living offspring were produced, which were later realized in succession in other species, but the success rate in clinic was still low, despite the great breakthrough.
Vitrification cryopreservation of porcine oocytes refers to a preservation technology for inhibiting metabolism activity of porcine oocytes in ultralow temperature environment, so that oocytes can be preserved for a long time without losing activity. The pig oocyte freezing preservation method mainly comprises a programmed freezing method and a vitrification freezing method. Experimental studies show that the vitrification freezing method of the porcine oocyte is superior to the programmed freezing method.
Common vitrification freezing methods are divided into a number of types: open elongated straws (OPS, open Pulled Straw), closed elongated straws (CPS, closed Pulled Straw), solid surface vitrification (SSV, solid Surface Vitrification), nylon Long Fa (Nylon-mesh), half wheat Guan Fa (Hemi-Straw), frozen ring (cryoop), and frozen cap (Cryotop) vitrification freezing. The Cryotop vitrification freezing method is the method with the best effect in the current freezing preservation technology, has the advantages of thin carrier and high cooling speed, the highest cooling speed reaches 60900K/min, and the slide glass is frozen by the smallest volume of the freezing liquid in the process of skilled operation, so that the slide glass can quickly cross the sensitive temperature area of the oocyte, and the survival rate of the oocyte after vitrification freezing is greatly improved.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a vitrification freezing method of porcine oocytes, which is a vitrification freezing technology with small damage to oocytes, low toxic and side effects and good freezing effect, and mature oocytes after thawing are successfully obtained, and the survival rate of the oocytes reaches 40.09%.
In order to solve the technical problems, the embodiment of the invention provides a vitrification freezing method of porcine oocytes, which comprises the following steps:
(1) Preparation of a liquid nitrogen box for pig oocyte freezing: filling liquid nitrogen into a liquid nitrogen box, fixing a 50ml centrifuge tube at one corner of the liquid nitrogen box, wherein the included angle between the centrifuge tube and the bottom of the liquid nitrogen box is 30-50 degrees, and the bottom of the centrifuge tube is provided with a liquid inlet for liquid nitrogen to flow into the centrifuge tube;
(2) Preparing in-vitro maturation liquid and operation liquid of pig oocytes:
(2-1) preparation of in vitro maturation liquid of pig oocytes: 10mM L-carnitine,1% (v/v) L-Glutamax,10% (v/v) PFF,10% (v/v) FBS,1.5% (v/v) PMSG,1.5% (v/v) hCG and 0.242% (v/v) gentamicin are dissolved in M-199 to prepare pig oocyte in vitro maturation liquid; filtering the in-vitro maturation liquid of the pig oocyte by using a filter with the caliber of 0.22 mu m, subpackaging by using a 1000 mu l/tube, and preserving at 4 ℃ for later use, wherein the effective period is one month;
(2-2) preparation of operating fluid:
T0：0.002m NaHCO 3 0.002M Pyruvic Acid (Na-salt), 0.016M HEPES (Na-salt), 0.009M HEPES (Acid), 1% (v/v) L-Glutamax dissolved in M-199;
HM:20% (v/v) FBS was dissolved in T0;
t2:2% (v/v) FBS was dissolved in T0;
SM:0.8M cross, 20% (v/v) FBS in M-199;
ES:10% (v/v) EG,10% (v/v) DMSO,16% (v/v) FBS dissolved in T0;
VS:20% (v/v) EG,20% (v/v) DMSO was dissolved in SM;
w1:0.267m sucrose,20% (v/v) FBS was dissolved in T0;
w2:0.16m cross, 20% (v/v) FBS in T0;
all the liquids are filtered and split-packed by a filter with the caliber of 0.22 mu m, 400 mu l/pipe is stored at the temperature of 4 ℃ for standby, and the effective period is one month;
(3) Manufacturing a freezing operation panel: two Nunc four-hole plates are adopted, one of the Nunc four-hole plates is a freezing disc and is marked as an F disc, and the liquid added in the corresponding 1-4 holes is F1-HM, F2-HM, F3-ES and F4-VS in sequence; the other Nunc four-hole plate is a thawing plate and is marked as a T plate, the liquid corresponding to 1 to 4 holes is sequentially T1-W1, T2-W1, T3-W2 and T4-HM, and the liquid quantity of each hole is 300 to 500 mu l;
(4) Oral straw for oocyte operation: the two ends of the filter are connected with transparent plastic pipes, one end of one transparent plastic pipe is inserted into the gun head, and one end of the other transparent plastic pipe is connected with the glass needle;
(5) Cryotop vector for pig oocyte freezing: one end of the Cryotop carrier is a handheld operating rod, the other end of the Cryotop carrier is a flat ultrathin and sheet Cryotop slide, and a protective sleeve is arranged on the outer side of the Cryotop slide;
(6) Selecting 2-6 mm transparent follicles from the frozen oocytes, uniformly coating at least 3 layers of cumulus oocyte complexes of cumulus granulosa cells, and in-vitro maturing the cumulus oocyte complexes in the L-IVM liquid prepared in the step (2-1) for 16-24 hours to obtain semi-matured COCs;
(7) Freezing and thawing pig oocyte Cryotop:
(7-1) transferring all COCs in the step (6) to the F1-HM by using the oral suction tube prepared in the step (4) through in-vitro culture for 16-24h, and then transferring all the COCs in the F1-HM to the F2-HM by replacing a clean glass needle with the same size;
(7-2) slowly sucking 3-5 COCs from the F2-HM by the mouth suction pipe prepared in the step (4) each time, transferring the COCs into the F3-ES, immediately transferring the COCs into the F4-VS in the same manner after 30s, and balancing for 20s; 3-5 COCs are contained in less than 1 mu l of VS liquid and are placed at the top end of the Cryotop slide in the step (5), and then the top end of the Cryotop slide is rapidly put into liquid nitrogen of the centrifuge tube in the step (1);
(7-3) implanting the tip of the glass needle connected to the straw into the hole bottom each time the COCs are transferred, slowly blowing out the COCs, avoiding the generation of bubbles and preventing the COCs from floating on the surface of the liquid; each transfer is guaranteed to be completed within 3 s; finally, when the liquid moves onto the slide, the liquid amount moved together with the COCs does not exceed 1 mu l; transferring COCs into F4-VS liquid until the liquid nitrogen is placed in the liquid nitrogen, wherein the process is strictly controlled within 1min;
(7-4) the Cryotop carrier loaded with COCs is quickly placed into a 50ml centrifuge tube, and the height of liquid nitrogen is 4-4.5 cm;
(7-5) before thawing, preparing a liquid nitrogen box and a T disc, preheating the T disc on a heat table at 38.5 ℃ for 30min, taking out a Cryotop carrier from liquid nitrogen by forceps each time, and putting a Cryotop slide with COCs into T1-W1, wherein the operation is controlled within 3 s; the thawed COCs were then transferred to T2-W1 and equilibrated for 1min; transferring into T3-W2, and balancing for 3min; finally, moving into the T4-HM; the liquid amount transferred each time is not more than 1 mu L, after thawing of 15-20 Cryotop carriers is completed, all COCs are transferred from T4-HM to L-IVM for 2 times of cleaning, thawing liquid is removed, and the COCs are transferred to IVM for Kong Zhongji continuous in-vitro culture;
(8) Evaluation after freezing of porcine oocytes: culturing the thawed COCs in the L-IVM solution for 26-30 hours; then, the COCs digest the cumulus granulosa cells by using 1mg/ml hyaluronidase, and the cumulus granulosa cells are blown for 20-25 s by a liquid-transferring gun; transferring the bare oocyte with the granulosa cells removed into the T2 prepared in the step (2-2), and observing under a microscope that the cytoplasm is smooth and complete and has a first polar body, and is a viable and mature oocyte.
In the step (1), the liquid nitrogen box is a self-made foam box, is a cuboid made of foam materials, has the outer diameter of 22.3cm, the inner diameter of 19.2cm, the outer diameter of 15.4cm, the inner diameter of 12.9cm, the outer height of 10.3cm and the inner height of 6.1cm, and a 50ml centrifuge tube is fixed at one corner by using an iron wire.
Wherein, in the step (3), the F plate is preheated on a hot table at 38.5 ℃ for 30min before the freezing operation.
In the step (4), the filter is a 0.22 mu m filter, the inner diameter of the transparent plastic tube is 3.0 mm, the gun head is a 200 mu l gun head, the inner diameter of the thicker end of the glass needle is 2.0 mm, the length is 4.5cm, the inner diameter of the thinner end is 0.3-0.4 mm, and the length is 5.0 cm.
In the step (5), the size of the hand-held operation lever is as follows: length 7.5 cm, width 2.0 mm, and thickness 2.0 mm; the dimensions of the sheet slide were: length 3.0 cm, width 60 μm and thickness 60 μm.
The technical scheme of the invention has the following beneficial effects:
1. mature oocytes after thawing are successfully obtained by the vitrification freezing method, and the survival rate of the oocytes reaches 40.09 percent.
2. The pig oocyte contains a large amount of lipid drops, is sensitive to low temperature and is easy to cause freezing damage. Fatty acids can be activated to fatty acyl-CoA via beta oxidation, but neither fatty acyl-CoA nor non-esterified fatty acids can enter the mitochondria directly through the inner mitochondrial membrane, but rather their transfer must be achieved by means of a small-molecule fatty acyl carrier L-carnitine. Thus, the addition of L-carnitine before and after freezing of COCs helps to promote normal lipid metabolism of oocytes before and after freezing.
Drawings
FIG. 1 is a schematic diagram of a liquid nitrogen box for freezing porcine oocytes;
FIG. 2 is a schematic diagram of the structures of the F disc and the T disc in the present invention;
FIG. 3 is a schematic view of the structure of the oral siphon;
FIG. 4 is a schematic structural diagram of a Cryotop vector of the present invention;
FIG. 5 shows the cytosolic, packed at least 3 layers of cumulus granulosa cells of the present invention and the sub-mature COCs cultured for 16-24 hours for freezing;
FIG. 6 shows mature oocytes cultured in vitro for a further 26-30h after thawing;
FIG. 7 shows the specific steps of the vitrification freezing and thawing of COCs;
FIG. 8 is a graph showing the amount of liquid carried by COCs as they move onto a Cryotop slide;
FIG. 9 shows the Bodipy immunofluorescence and average fluorescence intensity of oocytes in control, non-frozen and treated groups.
Reference numerals illustrate:
1. a liquid nitrogen box; 2. centrifuging tube; 4. liquid nitrogen; 5. an iron wire; 6. a liquid inlet hole; 7. f, a disc; 8. a T-plate; 9. a mouth suction tube; 10. a filter; 11. a transparent plastic tube; 12. a gun head; 13. a glass needle; 14. a Cryotop vector; 15. a hand-held operation lever; 16. a Cryotop slide; 17. a protective sleeve; 18. COCs; 19. cumulus oocyte complex of cumulus granulosa cells.
Detailed Description
In order to make the technical problems, technical solutions and advantages to be solved more apparent, the following detailed description will be given with reference to the accompanying drawings and specific embodiments.
The Cryotop vector of the invention is purchased from KITAZATO corporation of Japan; L-Glutamax, FBS were purchased from Gibco, USA, and the remaining reagents were purchased from Sigma, USA.
The invention provides a vitrification freezing method of porcine oocytes, which comprises the following steps:
(1) Preparation of a liquid nitrogen box for pig oocyte freezing: the liquid nitrogen box 1 is a self-made foam box, is a cuboid made of foam materials, has the outer diameter of 22.3cm, the inner diameter of 19.2cm, the outer diameter of 15.4cm, the inner diameter of 12.9cm, the outer height of 10.3cm and the inner height of 6.1cm, liquid nitrogen 4 is filled in the liquid nitrogen box 1, a 50ml centrifuge tube 2 is fixed at one corner of the liquid nitrogen box 1 by an iron wire 5, the included angle between the centrifuge tube 2 and the bottom of the liquid nitrogen box 1 is 30-50 degrees, and a liquid inlet 6 for allowing the liquid nitrogen 4 to flow into the centrifuge tube is formed in the bottom of the centrifuge tube 2; as shown in fig. 1.
(2) Preparing in-vitro maturation liquid and operation liquid of pig oocytes:
(2-1) preparation of in vitro maturation liquid of pig oocytes:
pig oocyte in vitro maturation solution (L-IVM) is the usual in vitro maturation solution (IVM) with the addition of 10mM L-carnitine: 10mM L-carnitine,1% (v/v) L-Glutamax,10% (v/v) PFF (Pig Follicle Fluid, porcine follicular fluid), 10% (v/v) FBS (Fetal Bovine Serum ), 1.5% (v/v) PMSG (1000 IU/ml), 1.5% (v/v) hCG (1000 IU/ml), 0.242% (v/v) Gentamicin (Gentamicin) dissolved in M-199 (Medium 199) to prepare porcine oocyte in vitro maturation liquid (L-IVM); filtering the in-vitro maturation liquid of the pig oocyte by using a filter with the caliber of 0.22 mu m, subpackaging by using a 1000 mu l/tube, and preserving at 4 ℃ for later use, wherein the effective period is one month;
(2-2) preparation of operating fluid:
T0：0.002m NaHCO 3 0.002M Pyruvic Acid (Na-salt), 0.016M HEPES (Na-salt), 0.009M HEPES (Acid), 1% (v/v) L-Glutamax dissolved in M-199;
HM:20% (v/v) FBS was dissolved in T0;
t2:2% (v/v) FBS was dissolved in T0;
SM:0.8M cross, 20% (v/v) FBS in M-199;
ES:10% (v/v) EG (ethylene glycol), 10% (v/v) DMSO (dimethyl sulfoxide), 16% (v/v) FBS dissolved in T0;
VS:20% (v/v) EG,20% (v/v) DMSO was dissolved in SM;
w1:0.267m sucrose,20% (v/v) FBS was dissolved in T0;
w2:0.16m cross, 20% (v/v) FBS in T0;
all the liquids are filtered and split-packed by a filter with the caliber of 0.22 mu m, 400 mu l/pipe is stored at the temperature of 4 ℃ for standby, and the effective period is one month.
(3) Manufacturing a freezing operation panel: two Nunc four-hole plates (Thermo company) are adopted, wherein one Nunc four-hole plate is a freezing disc and is marked as an F disc 7, and the liquid added into the corresponding 1-4 holes is F1-HM, F2-HM, F3-ES and F4-VS in sequence; the other Nunc four-hole plate is a thawing plate and is marked as a T plate 8, the liquid corresponding to 1-4 holes is sequentially T1-W1, T2-W1, T3-W2 and T4-HM, and the liquid quantity of each hole is 400 mu l; the F pan 7 was preheated on a hot table at 38.5 ℃ for 30min prior to the freezing operation. The structures of the F-disc and the T-disc are shown in fig. 2, wherein fig. 2 (a) is a schematic structural diagram of the F-disc, and fig. 2 (b) is a schematic structural diagram of the T-disc.
(4) The structure of the oral pipette 9 for oocyte manipulation is shown in FIG. 3: 0.22 The two ends of the [ mu ] m filter 10 are connected with transparent plastic pipes 11 with the inner diameter of 3.0 mm, one end of one transparent plastic pipe 11 is inserted into a 200 [ mu ] l gun head 12 (sterilization), one end of the other transparent plastic pipe 11 is connected with a self-made glass needle 13, the inner diameter of a thicker end 13-A of the glass needle 13 is 2.0 mm, the length is 4.5cm, the inner diameter of a thinner end 13-B is 0.3-0.4 mm, and the length is 5.0 cm. Fig. 3 (a) shows a schematic view of the structure of the oral suction tube, and fig. 3 (b) shows a schematic view of the structure of the glass needle.
(5) The structure of the Cryotop vector 14 for freezing porcine oocytes is shown in FIG. 4: one end of the Cryoop carrier 14 is a hand-held operating rod 15 with the length of 7.5 cm, the width of 2.0 mm and the thickness of 2.0 mm, the other end is a flat ultrathin sheet-shaped Cryoop slide 16, and the dimensions of the Cryoop slide 16 are 3.0 cm in length, 60 mu m in width and 60 mu m in thickness; the outside of the Cryotop slide 16 is provided with a protective sheath 17.
(6) Selecting 2-6 mm transparent follicles from the frozen oocytes, uniformly coating at least 3 layers of Cumulus Oocyte Complexes 19 (Cumulus-Oocyte Complexes, COCs; fig. 5A), and in-vitro maturing the Cumulus oocytes in the L-IVM solution prepared in the step (2-1) for 16-24 hours to obtain semi-matured COCs (fig. 5B);
(7) The steps of freezing and thawing the pig oocyte Cryotop are shown in figure 7,
(7-1) transferring all COCs in the step (6) to the F1-HM by using the mouth suction pipe 9 prepared in the step (4) through in-vitro culture for 16-24h, and then transferring all the COCs in the F1-HM to the F2-HM by replacing a clean glass needle 13 with the same size; the key point is that the new glass needle 13 is prevented from being stained with paraffin oil carried when COCs in the F1-HM are transferred for the first time, and the paraffin oil pollution in the F2-HM is avoided;
(7-2) slowly sucking 3-5 COCs from the F2-HM by the mouth suction pipe 9 prepared in the step (4) each time, transferring the COCs into the F3-ES, immediately transferring the COCs into the F4-VS in the same manner after 30s, and balancing for 20s; 3-5 COCs18 contained in less than 1 mu l of VS liquid are placed at the top end of the Cryoop slide 16 in the step (5), and then the top end of the Cryoop slide 16 is rapidly put into liquid nitrogen of the centrifuge tube 2 in the step (1);
(7-3) implanting the tip of the glass needle 13 connected to the mouth pipette 9 into the bottom of the hole each time COCs are transferred, slowly blowing out COCs, avoiding the generation of bubbles and preventing the COCs18 from floating on the surface of the liquid; each transfer is guaranteed to be completed within 3 s; finally, when the liquid moves onto the Cryotop slide 16, the amount of the liquid which moves together with the COCs does not exceed 1 mu l; transferring COCs into F4-VS liquid until the liquid nitrogen is placed in the liquid nitrogen, wherein the process is strictly controlled within 1min;
(7-4) the Cryotop carrier 14 loaded with COCs18 is rapidly placed into a 50ml centrifuge tube 2, and the height of liquid nitrogen is 4-4.5 cm; the Cryotop carrier 14 is prevented from floating due to excessive liquid nitrogen, and the freezing effect is reduced; if the centrifuge tubes are preserved for a long time, 15-20 Cryotop carriers 14 are put into each 50ml centrifuge tube 2, then the caps of the centrifuge tubes 2 are covered, and the centrifuge tubes are quickly transferred into a liquid nitrogen tank for storage;
(7-5) before thawing, preparing the liquid nitrogen box 1 and the T disc 8,T disc 8, preheating for 30min on a heat table at 38.5 ℃, taking out a Cryoop carrier 14 from liquid nitrogen by tweezers each time, and putting the Cryoop slide 16 with COCs18 into the T1-W1, wherein the operation is controlled within 3s, so that the loss of COCs due to overhead stay is avoided; the thawed COCs were then transferred to T2-W1 and equilibrated for 1min; transferring into T3-W2, and balancing for 3min; finally, moving into the T4-HM; the amount of liquid transferred each time is not more than 1 [ mu ] L (figure 8), after thawing of 15-20 Cryotop carriers 14 is completed, all COCs are transferred into L-IVM from T4-HM for 2 times, thawing liquid is removed, and transferred into IVM for Kong Zhongji continuous in-vitro culture;
(8) Evaluation after freezing of porcine oocytes: the thawed COCs are continuously cultured in the L-IVM solution for 26 to 30 hours (figure 6); then, the COCs digest the cumulus granulosa cells by using 1mg/ml hyaluronidase, and the cumulus granulosa cells are blown for 20-25 s by a liquid-transferring gun; the naked oocyte from which granulosa cells were removed was transferred into T2 (in vitro working fluid) prepared in step (2-2), and the cytoplasm was smooth and intact, with the first polar body, as a viable and mature oocyte, as observed under a microscope (FIG. 6).
A technique for vitrification freezing of porcine oocytes according to the present invention will be further described with reference to several examples.
Example 1: comparison of survival rates of COCs after thawing in control and treatment groups
COCs were collected, control group was matured in vitro in usual IVM solution, and treatment group was matured in vitro in L-IVM solution with 10mM L-carnitine added. Culturing the two groups of COCs in vitro for 16-24 hours, and freezing and thawing according to the step (7); then washing for 2 times in IVM liquid/L-IVM liquid respectively, removing thawing liquid, and transferring into respective original mature liquid (IVM/L-IVM) to continue in vitro culture for 26-30h under the same conditions; after maturation, the granulosa cells were removed by digestion with 1mg/ml hyaluronidase, and the survival rate of the two groups of oocytes was observed and recorded under a microscope, and the results are shown in Table 1, wherein the survival rate of the treated group is higher than that of the control group, indicating that the addition of L-carnitine to the IVM solution can increase the survival rate of the oocytes after freezing.
Example 2: effect of L-carnitine addition on frozen oocyte lactone droplets
COCs are divided into three groups: the COCs of the control group, the non-freezing group and the control group are matured in vitro in the common IVM liquid, the COCs of the treatment group are matured in vitro in the L-IVM liquid added with 10mM L-carnitine, the COCs of the control group and the COCs of the treatment group are cultured in vitro for 16-24 hours, and the COCs are frozen and thawed according to the step (7); then washing for 2 times in IVM liquid/L-IVM liquid respectively, removing thawing liquid, and transferring into respective original mature liquid (IVM/L-IVM) to continue in vitro culture for 26-30h under the same conditions; mature COCs were digested with 1mg/ml hyaluronidase and granulosa cells were removed, and M was selected from three groups of oocytes to remove the first polar bodyThe phase oocytes were stained with Bodipy 493/503 (Thermo Fisher, USA) lipid droplets. First washed 3 times with PB1 (1% (g/v) BSA (Bovine Serum Albumin) in PBS (Phosphate Buffered Saline)),5 min/time; transferring the oocyte into 4% paraformaldehyde, fixing at room temperature for 30min, and washing with PB1 for 3 times and 5 min/time; membrane permeation in 0.5% triton for 30min, pb1 wash 3 times, 5 min/time; 2. Mu.g/ml Bodipy was stained 1h, PB1 washed 3 times, 5 min/time in dark at 37 ℃; subsequently 10. Mu.g/ml Hoechst33342 was stained for 20min, washed 3 times with PB1 for 5 min/time, finally tabletted and photographed by observation with a laser confocal microscope, and the fluorescence intensity was analyzed by Image J software, as a result of which, as shown in FIG. 9B, although the fluorescence intensity of lipid droplets in oocytes of the treated group was significantly lower than that of the non-frozen group, it was significantly higher than that of the control group (P<0.05). Unlike the non-frozen group, which had small and dense droplets, the control group had significantly impaired droplets and the droplets grew to aggregate, but the treated group with L-carnitine had a similar distribution of droplets to the non-frozen group (FIG. 9A), indicating that L-carnitine protected the droplets during freezing.
Example 3: influence of details of the procedure on oocytes
During the freezing process, the details of the operation are very important, such as the amount of liquid at VS on the Cryotop slide 16. According to the experimental results (FIG. 8), the amount of liquid on the Cryotop slide 16 is preferably less than 1. Mu.l, and a liquid amount of more than 1. Mu.l increases the loss rate of oocytes and decreases the survival rate of oocytes, with higher liquid amounts leading to higher loss and apoptosis rates. Wherein FIG. 8 (a) shows the amount of liquid transferred into the Cryotop slide along with COCs <1 μl; FIG. 8 (b) shows that the amount of liquid transferred into the Cryotop slide together with COCs is 1-2. Mu.l; FIG. 8 (c) shows the amount of liquid transferred into the Cryotop slide along with COCs > 2. Mu.l.
The vitrification freezing method of the pig oocyte provided by the experiment is simple and effective, achieves the aim of freezing preservation, avoids damage to the oocyte caused by temperature reduction, can solve the obstacle of cross-regional germplasm resource communication, and protects local breeds of pigs.
While the foregoing is directed to the preferred embodiments of the present invention, it will be appreciated by those skilled in the art that various modifications and adaptations can be made without departing from the principles of the present invention, and such modifications and adaptations are intended to be comprehended within the scope of the present invention.
Claims (5)
1. A method for vitrification and freezing of porcine oocytes, which is characterized by comprising the following steps:
(1) Preparation of a liquid nitrogen box for pig oocyte freezing: filling liquid nitrogen into a liquid nitrogen box, fixing a 50ml centrifuge tube at one corner of the liquid nitrogen box, wherein the included angle between the centrifuge tube and the bottom of the liquid nitrogen box is 30-50 degrees, and the bottom of the centrifuge tube is provided with a liquid inlet for liquid nitrogen to flow into the centrifuge tube;
(2) Preparing in-vitro maturation liquid and operation liquid of pig oocytes:
(2-1) preparation of in vitro maturation liquid of pig oocytes: 10mM L-carnitine,1% (v/v) L-glutamine, 10% (v/v) follicular fluid, 10% (v/v) fetal bovine serum, 1.5% (v/v) pregnant mare serum gonadotropin, 1.5% (v/v) human chorionic gonadotropin, 0.242% (v/v) gentamicin dissolved in M-199, and the porcine oocyte in vitro maturation liquid is prepared; filtering the in-vitro maturation liquid of the pig oocyte by using a filter with the caliber of 0.22 mu m, subpackaging by using a 1000 mu l/tube, and preserving at 4 ℃ for later use, wherein the effective period is one month;
(2-2) preparation of operating fluid:
t0:0.002M sodium bicarbonate, 0.002M pyruvic acid, 0.016M N-2-hydroxyethylpiperazine-N '-2-ethanesulfonic acid, 0.009M N-2-hydroxyethylpiperazine-N' -2-ethanesulfonic acid, 1% (v/v) L-glutamine dissolved in M-199;
HM:20% (v/v) fetal bovine serum was dissolved in T0;
t2:2% (v/v) fetal bovine serum was dissolved in T0;
SM:0.8M sucrose,20% (v/v) fetal bovine serum in M-199;
ES:10% (v/v) ethylene glycol, 10% (v/v) dimethyl sulfoxide, 16% (v/v) fetal bovine serum was dissolved in T0;
VS:20% (v/v) ethylene glycol, 20% (v/v) dimethyl sulfoxide was dissolved in SM;
w1:0.267M sucrose,20% (v/v) fetal bovine serum in T0;
w2:0.16M sucrose,20% (v/v) fetal bovine serum in T0;
all the liquids are filtered and split-packed by a filter with the caliber of 0.22 mu m, 400 mu l/pipe is stored at the temperature of 4 ℃ for standby, and the effective period is one month;
(3) Manufacturing a freezing operation panel: two Nunc four-hole plates are adopted, one of the Nunc four-hole plates is a freezing disc and is marked as an F disc, and the liquid added in the corresponding 1-4 holes is F1-HM, F2-HM, F3-ES and F4-VS in sequence; the other Nunc four-hole plate is a thawing plate and is marked as a T plate, the liquid corresponding to 1 to 4 holes is sequentially T1-W1, T2-W1, T3-W2 and T4-HM, and the liquid quantity of each hole is 300 to 500 mu l;
(4) Oral straw for oocyte operation: the two ends of the filter are connected with transparent plastic pipes, one end of one transparent plastic pipe is inserted into the gun head, and one end of the other transparent plastic pipe is connected with the glass needle;
(5) Cryotop vector for pig oocyte freezing: one end of the Cryotop carrier is a handheld operating rod, the other end of the Cryotop carrier is a flat ultrathin and sheet Cryotop slide, and a protective sleeve is arranged on the outer side of the Cryotop slide;
(6) Selecting 2-6 mm transparent follicles from the frozen oocytes, uniformly coating at least 3 layers of cumulus oocyte complexes of cumulus granulosa cells, and in-vitro maturing the cumulus oocyte complexes in the L-IVM liquid prepared in the step (2-1) for 16-24 hours to obtain semi-matured COCs;
(7) Freezing and thawing pig oocyte Cryotop:
(7-1) transferring all COCs in the step (6) to the F1-HM by using the oral suction tube prepared in the step (4) through in-vitro culture for 16-24h, and then transferring all the COCs in the F1-HM to the F2-HM by replacing a clean glass needle with the same size;
(7-2) slowly sucking 3-5 COCs from the F2-HM by the mouth suction pipe prepared in the step (4) each time, transferring the COCs into the F3-ES, immediately transferring the COCs into the F4-VS in the same manner after 30s, and balancing for 20s; 3-5 COCs are contained in less than 1 mu l of VS liquid and are placed at the top end of the Cryotop slide in the step (5), and then the top end of the Cryotop slide is rapidly put into liquid nitrogen of the centrifuge tube in the step (1);
(7-3) implanting the tip of the glass needle connected to the straw into the hole bottom each time the COCs are transferred, slowly blowing out the COCs, avoiding the generation of bubbles and preventing the COCs from floating on the surface of the liquid; each transfer is guaranteed to be completed within 3 s; finally, when the liquid moves onto the slide, the liquid amount moved together with the COCs does not exceed 1 mu l; transferring COCs into F4-VS liquid until the liquid nitrogen is placed in the liquid nitrogen, wherein the process is strictly controlled within 1min;
(7-4) the Cryotop carrier loaded with COCs is quickly placed into a 50ml centrifuge tube, and the height of liquid nitrogen is 4-4.5 cm;
(7-5) before thawing, preparing a liquid nitrogen box and a T disc, preheating the T disc on a heat table at 38.5 ℃ for 30min, taking out a Cryotop carrier from liquid nitrogen by forceps each time, and putting a Cryotop slide with COCs into T1-W1, wherein the operation is controlled within 3 s; the thawed COCs were then transferred to T2-W1 and equilibrated for 1min; transferring into T3-W2, and balancing for 3min; finally, moving into the T4-HM; the liquid amount transferred each time is not more than 1 mu L, after thawing of 15-20 Cryotop carriers is completed, all COCs are transferred from T4-HM to L-IVM for 2 times of cleaning, thawing liquid is removed, and the COCs are transferred to IVM for Kong Zhongji continuous in-vitro culture;
(8) Evaluation after freezing of porcine oocytes: culturing the thawed COCs in the L-IVM solution for 26-30 hours; then, the COCs digest the cumulus granulosa cells by using 1mg/ml hyaluronidase, and the cumulus granulosa cells are blown for 20-25 s by a liquid-transferring gun; transferring the bare oocyte with the granulosa cells removed into the T2 prepared in the step (2-2), and observing under a microscope that the cytoplasm is smooth and complete and has a first polar body, and is a viable and mature oocyte.
2. The method for vitrification freezing of porcine oocytes according to claim 1, wherein in the step (1), the liquid nitrogen box is a self-made foam box, which is a cuboid made of foam material, and has an outer diameter of 22.3cm, an inner diameter of 19.2cm, an outer diameter of 15.4cm, an inner diameter of 12.9cm, an outer height of 10.3cm and an inner height of 6.1cm, wherein a 50ml centrifuge tube is fixed at one corner by iron wires.
3. The method for vitrification freezing of porcine oocytes according to claim 1, wherein in the step (3), the F-disc is preheated on a hot stage at 38.5 ℃ for 30min before the freezing operation.
4. The method for vitrification and freezing of porcine oocytes according to claim 1, wherein in the step (4), the filter is a 0.22 μm filter, the inner diameter of the transparent plastic tube is 3.0 mm, the gun head is 200 μl gun head, the inner diameter of the thicker end of the glass needle is 2.0 mm, the length is 4.5cm, the inner diameter of the thinner end is 0.3-0.4 mm, and the length is 5.0 cm.
5. The method for vitrification freezing of porcine oocytes according to claim 1, wherein in the step (5), the size of the hand-held lever is: length 7.5 cm, width 2.0 mm, and thickness 2.0 mm; the dimensions of the sheet slide were: length 3.0 cm, width 60 μm and thickness 60 μm.
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