CN108707578B - Pig monose embryo in vitro culture method - Google Patents

Abstract

The invention discloses a pig single embryo in vitro culture method. Belongs to the field of agriculture, animal husbandry and veterinary medicine. Specifically, feeder layer cell microdroplets and single embryos are adopted for co-culture, a good development result is obtained, the development process of single embryo culture is consistent with that of traditional multi-embryo culture, and the single embryos with zona pellucida attached or zona pellucida removed can obtain parthenogenetic development blastula of the pigs for 7 days. The feeder layer cell microdroplet provided by the invention is simple to prepare, can provide reliable raw materials for researching the development of a single embryo according to the method provided by the invention, and can completely meet the requirements of tracking observation and further research on the development process of the single embryo of the pig. The single embryo culture method provided by the invention can be widely applied to single embryo culture of various species.

Description

Pig monose embryo in vitro culture method

Technical Field

The invention belongs to the field of agriculture, animal husbandry and veterinary medicine, and particularly relates to an in vitro culture method of a swine parthenogenetic activated embryo single embryo.

Background

In recent years, in vitro embryo production is widely used for producing economically valuable animals, disease models, bioreactors and the like, and meanwhile, the development of animal husbandry is greatly promoted by the application of in vitro fertilization technology (IVF), single sperm injection technology (ICSI), embryo transfer technology, embryo segmentation technology, embryonic stem cell engineering and the like, however, in the development and application of the new technologies, the development and application of the new technologies are greatly limited by the low development capability and early embryonic development retardation of in vitro embryo production. Although many studies have been made to improve the development ability of embryos by adding or reducing factors to the culture medium of in vitro culture of early mammalian embryos, the quality and blastocyst development rate of embryos produced in vitro are still far from those of embryos developed in vivo, which requires further research.

With the rapid development of the fields of social economy, animal husbandry and the like, more and better animal embryos need to be prepared in vitro urgently, however, in the actual production process, the embryo retardation of in vitro development is particularly serious at a certain stage, and a large amount of resources are wasted.

The phenomenon of in vitro development retardation of mammalian early embryos refers to the phenomenon that embryos cannot continue to develop due to the occurrence of blocking in the process of fertilized egg development from zygote stage to blastula stage, the time of early embryo development retardation varies with species, but is often related to the time of development regulation transition, goats and sheep both occur at 8-16 cell stages of embryos, human embryos are generally easy to block at 4-8 cell stages of embryos in the in vitro development process, and pig embryos are severely blocked at 4 cell stages in the in vitro development process. Therefore, the molecular mechanism involved in the development of early embryonic development arrest in mammals is one of the hot spots in current reproductive and developmental biology research.

The embryo development retardation phenomenon is researched by firstly obtaining a research object and ensuring that the research object exists independently. Currently, research aiming at single embryo mainly focuses on mouse fertilized eggs and embryos at all development stages, and the mouse embryos are mainly obtained by in vivo washing, so that the mouse embryos are relatively simple to obtain and low in cost. And aiming at the in vitro culture of the mouse single embryo, the calcium alginate gel embedding culture method is mainly selected after the zona pellucida is removed. Because of the species difference, the embryo size, the embryo in vitro culture conditions (including culture medium, culture temperature, etc.) and the culture requirements are different, the mouse single embryo in vitro culture method is not suitable for the single embryo in vitro culture of the large mammal, namely the pig.

Therefore, it is urgently needed to establish a single embryo in vitro culture method suitable for pigs so as to meet the requirements of tracking and observing the development process of single embryos of the pigs and further researching the development process of the single embryos.

Disclosure of Invention

In order to solve the problems in the prior art, the method for co-culturing the feeder layer cell microdroplets and the single embryo is used for in-vitro culture of the single embryo of the pig, so that a better development result is obtained, and the requirements of tracking observation and further research on the development process of the single embryo of the pig can be completely met.

The invention achieves the purpose through the following technical scheme:

in a first aspect, the invention provides a method for culturing pig single embryos in vitro, which comprises the following steps:

1) preparing mature oocytes in vitro of pigs:

collecting ovaries of sows; washing ovarian tissue with 75% alcohol for 1min, cleaning with sterilized and preheated normal saline for 3 times, extracting ovarian follicle with diameter of 3-8mm with sterile syringe equipped with standard 20-gauge needle (containing small amount of balanced DPBS), injecting the extractive solution into 50mL pointed-bottom centrifuge tube, and placing into the pointed-bottom centrifuge tube in 39 deg.C water bath; cumulus-oocyte complex (COC)_S) After precipitation, eggs were examined under a stereomicroscope after 2 washes with DPBS (purchased from GIBCO). Picking up COCs which are normal in shape, uniform in cytoplasm and wrapped by at least 3 layers of granular cells, washing the detected COCs for 3 times by using DPBS (DPBS) containing 5% Fetal Bovine Serum (FBS), then washing the COCs for 3 times by using in-vitro maturation culture solution, and transferring the COCs into a five-hole cell culture plate which contains 500 mu L of in-vitro maturation culture solution and is covered with paraffin oil; placing 100-150 COCs per drop; the liquid is pre-treated with CO₂Balancing in an incubator for more than 2 hours; culturing in vitro at 39 deg.C for 42-44 hr with 5% CO₂Saturated humidity;

the in vitro maturation culture solution is TCM-199(GIBCO) +0.1% polyvinyl alcohol +3.05mM glucose +0.91mM sodium pyruvate +0.57mM L-cysteine +100IU/mL penicillin + 100. mu.g/mL streptomycin +10% (V/V) follicular fluid +10IU/mL PMSG (Ningbo hormone products factory) +10IU/mL hCG (Ningbo hormone products factory);

2) parthenogenetic activation of porcine in vitro mature oocytes:

COCs matured in vitro for 42-44h were transferred into DPBS containing 0.1% hyaluronidase for digestion and gently blown with a pipette gun repeatedly to remove cumulus cells, washed 3 times with pre-warmed activation solution, placed in a fusion tank with activation solution, electrically activated with BTX-2001 fusion instrument with activation parameters: 1 direct current pulse of 30 mus and 1.1 kv/cm; the activated oocyte is washed for three times by embryo culture solution NCSU-23 preheated in advance for standby;

the formula of the activating liquid comprises: 0.25mol/L mannitol, 0.5mol/L calcium chloride, 0.5mol/L magnesium sulfate, 0.5mol/L HEPES and 0.01% PVA;

3) co-culture feeder layer cell droplet preparation:

(1) preparation of microdroplets of oviduct epithelial cell feeder layer

A. Isolation and primary culture of oviduct epithelial cells: putting a sow oviduct after obtaining an oviduct of a sow, adding sterile physiological saline added with double antibodies at 30-37 ℃ into the sterile physiological saline to be brought back to a laboratory within 2h, shearing redundant oviduct membranes by using sterile scissors, shearing the oviduct into small sections with the length of 2cm, washing for 3 times by using sterile physiological saline added with double antibodies at 35 ℃, putting the small sections into a disposable plastic dish with the diameter of 35mm, flushing epithelial cells while observing under a stereoscopic microscope, clamping a fracture on one side of the oviduct by using forceps of a clock and a watch in the operation process, sucking pancreatin with the working concentration of 0.25 percent by using a disposable syringe with the volume of 1 ml in the right hand, directly and slowly injecting from the fracture of the oviduct clamped by the forceps to flush the oviduct epithelial cells, collecting pancreatin the dish after repeating for 2-3 times, quickly adding DMEM containing 10 percent of FBS with the same volume to stop digestion, and avoiding excessive digestion, the cell is dead, and the time of pancreatin in the oviduct is controlled within 5min so as to ensure the purity of the oviduct epithelial cell. Centrifuging the collected liquid at 3000r/min for 5min, discarding the supernatant, adding DMEM containing 10% FBS, repeatedly centrifuging for 2 times, discarding the supernatant, and placing the obtained precipitate into a 6-well plate containing 2 ml of DMEM containing 10% FBS for culture; after culturing for 3 days, replacing a fresh culture medium, and digesting for later use when the cells grow over the bottom of the 6-pore plate;

B. feeder layer co-culture cell droplet preparation: digesting oviduct epithelial cells overgrowing with 6-well plate with 0.25% pancreatin 48h before co-culture for 2min, adding DMEM containing 10% FBS to stop digestion, gently blowing into single cell suspension with 1 ml pipette, and collecting cellsDensity adjusted to 10⁴The number/ml of the cells in the cell suspension is ensured to exist uniformly, the cell suspension is used for preparing the co-culture feeder layer single embryo culture microdroplet, namely, a pipettor with the measuring range of 10 mu L is used for sucking 10 mu L of cell suspension, and the cell microdroplet is made on a disposable capsule with the diameter of 35 mm; each droplet size 10. mu.L of overlay paraffin oil, placed at 39 ℃ in 5% CO₂Culturing in an incubator with saturated humidity, and culturing for 48h for cell co-culture;

alternatively, the first and second electrodes may be,

(2) cumulus granulosa cell feeder layer droplet preparation:

placing the cultured mature oocyte in a maturation solution, repeatedly blowing and beating cumulus cells except the oocyte by using a liquid transfer gun, completely blowing and picking up the oocyte, keeping the remaining cumulus granular cells in the solution, and adjusting the cell density to 10⁴The number/ml of the cells in the cell suspension is ensured to exist uniformly, the cell suspension is used for preparing the co-culture feeder layer single embryo culture microdroplet, namely, a pipettor with the measuring range of 10 mu L is used for sucking 10 mu L of cell suspension, and the cell microdroplet is made on a disposable capsule with the diameter of 35 mm; each droplet size 10. mu.L of overlay paraffin oil, placed at 39 ℃ in 5% CO₂Culturing in an incubator with saturated humidity, and culturing for 48h for cell co-culture;

alternatively, the first and second electrodes may be,

(3) porcine fetal fibroblast feeder layer droplet preparation

A. Preparation of porcine fetal fibroblasts: the specific operation method refers to the method disclosed in ZL201310381274.5 Hubei white pig fibroblast line;

B. feeder layer co-culture cell droplet preparation: digesting pig fetal fibroblast overgrown with 6-well plate with 0.25% pancreatin 48h before co-culture for 2min, adding DMEM containing 10% FBS to stop digestion, gently blowing into single cell suspension with 1 ml pipette, and adjusting cell density to 10⁴The number/ml of the cells in the cell suspension is ensured to exist uniformly, the cell suspension is used for preparing the co-culture feeder layer single embryo culture microdroplet, namely, a pipettor with the measuring range of 10 mu L is used for sucking 10 mu L of cell suspension, and the cell microdroplet is made on a disposable capsule with the diameter of 35 mm; each droplet size 10 μ L covering stoneWax oil, 5% CO at 39 deg.C₂Culturing in an incubator with saturated humidity, and culturing for 48h for cell co-culture;

4) co-culturing single embryo and feeder layer cells:

(1) co-culture of oocyte and feeder layer cell with zona pellucida attachment

Changing a cell culture medium DMEM in the microdroplets into a fresh embryo culture medium NCSU-23 2 hours before the oocytes are placed into the microdroplets for later use;

selecting in-vitro mature oocytes with first polar bodies and uniform cytoplasm obtained in the step 2) to be in an embryo culture medium NCSU-23 balanced for 2 hours in advance, washing for 3 times, and then putting the oocytes into the co-culture feeder layer single-cell microdroplets prepared in the step 3) for co-culture, wherein 1 oocyte is put in each microdroplet; carrying out half-amount liquid change within 48 h;

alternatively, the first and second electrodes may be,

(2) co-culture of zona pellucida-removed oocytes and feeder cells

Changing a cell culture medium DMEM in the microdroplets into a fresh embryo culture medium NCSU-23 2 hours before the oocytes are placed into the microdroplets for later use;

after in vitro mature oocytes are parthenogenetically activated, selecting in vitro mature oocytes with a first polar body, uniform cytoplasm and complete cell membranes, digesting the in vitro mature oocytes with pronase with the working concentration of 0.25% to remove zona pellucida, washing the zona pellucida-free oocytes in an embryo culture medium NCSU-23 balanced for 2 hours in advance for 3 times, placing the zona pellucida-free oocytes into single embryo culture microdroplets of the co-culture feeder layer prepared in the step 3) for co-culture, placing 1 oocyte in each microdroplet, and performing half-amount liquid change for 48 hours;

5) obtaining the parthenogenetic blastocysts of the pigs in 7 days: and (4) counting the embryo cleavage rate in 36h, observing the development condition of the embryo under a microscope after co-culture for 7d, and counting the blastocyst development rate.

Preferably, the single embryo co-cultured in step 4) of the above method is any one of an in vitro fertilized embryo, a single sperm injection embryo, and a somatic cell nuclear transfer embryo.

The invention has the following advantages:

the method for co-culturing the feeder layer cell microdroplets and the single embryos is used for in-vitro culture of the single embryos of the pigs for the first time. The better development result is obtained, the development process of single embryo culture is consistent with that of the traditional multi-embryo culture, and the parthenogenetic development blastocyst of the pig can be obtained 7 days from the single embryo with the zona pellucida attached or removed. The feeder layer cell microdroplet provided by the invention is simple to prepare, can provide reliable raw materials for researching the development of a single embryo according to the method provided by the invention, and can completely meet the requirements of tracking observation and further research on the development process of the single embryo of the pig. The single embryo culture method provided by the invention can be widely applied to single embryo culture of various species.

Drawings

FIG. 1 is a schematic representation of porcine oviduct epithelial cells in accordance with the present invention;

FIG. 2 is a schematic representation of porcine cumulus granulosa cells of the present invention;

FIG. 3 is a schematic representation of porcine fetal fibroblasts of the present invention;

FIG. 4 is a schematic representation of feeder cells co-cultured single embryo developmental blastocysts according to the invention;

A. the oocyte attached with the zona pellucida develops to a blastocyst; B. the oocyte with the zona pellucida removed develops to a blastocyst;

Detailed Description

The features and advantages of the present invention will be further understood from the following detailed description taken in conjunction with the accompanying drawings. The examples provided are merely illustrative of the method of the present invention and do not limit the remainder of the disclosure in any way. [ example 1 ] preparation of oocytes matured in vitro in pigs

After the ovaries of the sows are collected in a grain and meat food processing factory in Wuhan City, the ovaries are immediately put into sterilized normal saline with 35 ℃ added with double antibiotics for 2 to 4 hours and then sent back to a laboratory. Ovarian tissue is washed with 75% ethanol for 1min, washed with sterilized and preheated physiological saline for 3 times, and then follicles with a diameter of 3-8mm on the surface of the ovary are aspirated by a sterile syringe equipped with a standard 20-gauge needle (containing a small amount of equilibrated DPBS), and the aspirated solution is injected into a 50mL conical centrifuge tube and placed in a conical centrifuge tube in a 39 ℃ water bath. Cumulus-oocyte complex (COC)_S) After precipitation, useDpbs (gibco) was washed 2 times before egg examination under a stereomicroscope. CoCs that are morphologically normal, cytosolically homogeneous and have at least 3 granulosa cell envelopes were picked up, washed 3 times with DPBS containing 5% Fetal Bovine Serum (FBS), then 3 times with maturation medium, and transferred to five-well cell culture plates containing 500. mu.L of maturation medium and covered with paraffin oil. 100-150 COCs are put in each drop. The liquid is pre-treated with CO₂And balancing in the incubator for more than 2 hours. Culturing in vitro at 39 deg.C for 42-44 hr with 5% CO₂And saturation humidity.

The in vitro maturation medium was TCM-199(GIBCO) +0.1% polyvinyl alcohol +3.05mM glucose +0.91mM sodium pyruvate +0.57mM L-cysteine +100IU/mL penicillin + 100. mu.g/mL streptomycin +10% (V/V) follicular fluid +10IU/mL PMSG (Ningbo hormone products works) +10IU/mL hCG (Ningbo hormone products works).

[ example 2 ] parthenogenetic activation of oocytes matured in vitro in swine

COCs matured in vitro for 42-44h were transferred into DPBS containing 0.1% hyaluronidase for digestion and gently blown with a pipette gun repeatedly to remove cumulus cells, washed 3 times with pre-warmed activation solution, placed in a fusion tank with activation solution, electrically activated with BTX-2001 fusion instrument with activation parameters: 30 μ s, 1.1kv/cm, 1 direct current pulse. The activated oocytes were washed three times with pre-warmed embryo culture solution NCSU-23 and then kept ready for use.

The formula of the activating liquid comprises: 0.25mol/L mannitol +0.5mol/L calcium chloride +0.5mol/L magnesium sulfate +0.5mol/LHEPES +0.01% PVA.

Example 3 Co-culture feeder layer cell droplet preparation

(1) Preparation of microdroplets of oviduct epithelial cell feeder layer

A. Isolation and primary culture of oviduct epithelial cells: putting a sow oviduct after obtaining an oviduct of a sow, adding sterile physiological saline added with double antibodies at 30-37 ℃ into the sterile physiological saline to be brought back to a laboratory within 2h, shearing redundant oviduct membranes by using sterile scissors, shearing the oviduct into small sections with the length of 2cm, washing for 3 times by using sterile physiological saline added with double antibodies at 35 ℃, putting the small sections into a disposable plastic dish with the diameter of 35mm, flushing epithelial cells while observing under a body type microscope, clamping a fracture on one side of the oviduct by using a pair of tweezers in the operation process of holding a watch by the left hand, directly and slowly injecting pancreatin the working concentration of 0.25 percent from the fracture of the oviduct clamped by using a pair of tweezers by using a 1 ml disposable syringe by the right hand to flush the oviduct epithelial cells, collecting pancreatin the dish after repeating for 2-3 times, quickly adding DMEM containing 10 percent of the pancreatin the same volume to stop digestion, and avoiding excessive digestion, the cell is dead, and the time of pancreatin in the oviduct is controlled within 5min so as to ensure the purity of the oviduct epithelial cell. The collected liquid was centrifuged at 3000r/min for 5min, the supernatant was discarded, DMEM containing 10% FBS was added thereto and centrifugation was repeated 2 times to discard the supernatant, and the resulting precipitate was cultured in 6-well plates containing 2 ml of DMEM containing 10% FBS. After 3 days of culture, the fresh culture medium is replaced, and the cells can be digested for later use when growing over the bottom of the 6-well plate.

B. Feeder layer co-culture cell droplet preparation: digesting oviduct epithelial cells overgrowing with 6-well plate with 0.25% pancreatin 48h before co-culture for 2min, adding DMEM containing 10% FBS to stop digestion, gently blowing into single cell suspension with 1 ml pipette, and adjusting cell density to 10⁴One/ml and ensuring that the cells are uniformly present in the cell suspension, using this cell suspension to prepare CO-cultured feeder layer single embryo culture droplets, i.e., 10. mu.L of cell suspension is aspirated by a pipette measuring 10. mu.L, and cell droplets are made on disposable 35mm dishes, each 10. mu.L, covered with paraffin oil, placed at 39 ℃ and 5% CO₂Culturing in an incubator with saturated humidity, and culturing for 48h for cell co-culture.

(2) Cumulus granulosa cell feeder layer droplet preparation: placing the cultured mature oocyte in a maturation solution, repeatedly blowing and beating cumulus cells except the oocyte by using a liquid transfer gun, completely blowing and picking up the oocyte, keeping the remaining cumulus granular cells in the solution, and adjusting the cell density to 10⁴One/ml and ensuring that the cells are present uniformly in the cell suspension, using this cell suspension to prepare co-cultured feeder layer single embryo culture droplets 10. mu.L of cell suspension is aspirated using a pipette measuring 10. mu.L, and cell droplets are made on disposable 35mm dishes, each 10. mu.L, covered with paraffin oil, placed at 39 deg.C，5％CO₂Culturing in an incubator with saturated humidity, and culturing for 48h for cell co-culture.

(3) Porcine fetal fibroblast feeder layer droplet preparation

A. Preparation of porcine fetal fibroblasts: the specific operation method refers to the preparation method disclosed in Chinese patent Hubei white pig fibroblast line.

B. Feeder layer co-culture cell droplet preparation: digesting pig fetal fibroblast overgrown with 6-well plate with 0.25% pancreatin 48h before co-culture for 2min, adding DMEM containing 10% FBS to stop digestion, gently blowing into single cell suspension with 1 ml pipette, and adjusting cell density to 10⁴And ensuring that the cells exist uniformly in the cell suspension, and preparing a co-culture feeder layer single embryo culture microdroplet by using the cell suspension, namely sucking 10 mu L of cell suspension by using a pipette with the range of 10 mu L and making the cell microdroplet on a disposable capsule with the diameter of 35 mm. Each droplet size 10. mu.L of overlay paraffin oil, placed at 39 ℃ in 5% CO₂Culturing in an incubator with saturated humidity, and culturing for 48h for cell co-culture.

Example 4 Co-culture of cells

(1) Co-culture of oocyte and feeder layer cell with zona pellucida attachment

The cell culture medium DMEM in the microdroplets was replaced with fresh embryo culture medium NCSU-23 2h before the oocytes were placed in the microdroplets for future use.

After parthenogenetic activation of the in vitro mature oocytes, the in vitro mature oocytes with the first polar body, uniform cytoplasm and complete cell membranes are selected to be washed for 3 times in an embryo culture medium NCSU-23 balanced for 2 hours in advance and then placed into the single embryo culture microdroplets of the co-culture feeder layer prepared in the embodiment 3 for co-culture, and 1 oocyte is placed in each microdroplet. And half amount of liquid change is carried out for 48 hours. And (5) counting the embryo cleavage rate at 36h, and counting the blastocyst development rate at 7 d.

(2) The zona pellucida-removed oocytes were co-cultured with feeder cells.

The cell culture medium DMEM in the microdroplets was replaced with fresh embryo culture medium NCSU-23 2h before the oocytes were placed in the microdroplets for future use.

After parthenogenetic activation of the in vitro mature oocytes, the in vitro mature oocytes with the first polar body, uniform cytoplasm and complete cell membranes are selected and digested by pronase with the working concentration of 0.25% to remove the zona pellucida, the oocytes without the zona pellucida are washed for 3 times in an embryo culture medium NCSU-23 balanced for 2 hours in advance and then are placed into the co-culture feeder layer single embryo culture microdroplets prepared in the embodiment 3 for co-culture, and each microdroplet is placed with 1 oocyte. And half amount of liquid change is carried out for 48 hours. And (5) counting the embryo cleavage rate at 36h, and counting the blastocyst development rate at 7 d.

Example 5 Single embryo in vitro culture experiment Using conventional droplet culture method

In the experiment, microdroplets are prepared on a 35mm disposable cuvette for single embryo in-vitro culture, and a control experiment is mainly carried out: a: droplet sizes are divided into three groups: 5. mu.L/drop, 10. mu.L/drop, 50. mu.L/drop, 100. mu.L/drop; b: in vitro culture of single embryos using different media on the basis of droplet size groupings: firstly, a fresh pig embryo culture medium NCSU-23 is adopted to carry out in-vitro culture of single embryos; directly carrying out single embryo in-vitro culture on NCSU-23 cultured with blastocysts in the five-hole plate; ③ taking 25 percent of NCSU-23 which has cultured blastocyst in the five-hole plate and adding 75 percent of fresh NCSU-23 to directly carry out single embryo in vitro culture; taking 50% of NCSU-23 which has cultured blastocyst in the five-hole plate and adding 50% of fresh NCSU-23 to directly carry out single embryo in vitro culture; taking 75% NCSU-23 with blastocyst cultured in five-hole plate and adding 25% fresh NCSU-23 to directly perform single embryo in vitro culture; sixthly, adding 10 percent FBS into the fresh embryo culture medium NCSU-23; adding 1% of essential amino acid and 0.5% of non-essential amino acid into fresh NCSU-23; eighthly, adding 10 percent FBS into the fresh TCM 199; ninthly fresh TCM199 with 1% essential amino acids and 0.5% non-essential amino acids added.

Each experiment was repeated at least three times, and each control group had no less than 100 embryos.

As a result: the microdroplet size is 10 mu L/drop, the culture effect is better by adding 10% FBS into TCM199, the embryo cleavage rate is 60%, the embryo develops to 10% of 16 cells, no morula and blastocyst exist, and the embryo is mainly blocked at the 4-cell stage. When NCSU-23 is used for culture, the cells develop to 8 cell death, and the cleavage rate is 50%.

Example 6 in vitro culture of Single embryos Using traditional Microdrop method with feeder cells

In this experiment, feeder layer droplets were prepared on 35mm disposable dishes for in vitro culture of single embryos, with droplet size of 10. mu.L/droplet. The following control experiments were mainly performed: each feeder layer cell was replaced with medium in B. A: feeder layer cells including cumulus granular cells, porcine fetal fibroblasts, and oviduct epithelial cells; b: the culture medium comprises the following components: firstly, a fresh pig embryo culture medium NCSU-23 is adopted to carry out in-vitro culture of single embryos; directly carrying out single embryo in-vitro culture on NCSU-23 cultured with blastocysts in the five-hole plate; ③ taking 25 percent of NCSU-23 which has cultured blastocyst in the five-hole plate and adding 75 percent of fresh NCSU-23 to directly carry out single embryo in vitro culture; taking 50% of NCSU-23 which has cultured blastocyst in the five-hole plate and adding 50% of fresh NCSU-23 to directly carry out single embryo in vitro culture; taking 75% NCSU-23 with blastocyst cultured in five-hole plate and adding 25% fresh NCSU-23 to directly perform single embryo in vitro culture; sixthly, adding 10 percent FBS into the fresh embryo culture medium NCSU-23; adding 1% of essential amino acid and 0.5% of non-essential amino acid into fresh NCSU-23; eighthly, adding 10 percent FBS into the fresh TCM 199; ninthly fresh TCM199 with 1% essential amino acids and 0.5% non-essential amino acids added.

Each experiment was repeated at least three times, and each control group had no less than 100 embryos.

As a result: after the feeder layer cells are added, the in vitro culture effect of the single embryo is better than that of the cells without the feeder layer cells. Compared with the traditional method of TCM-199, the method has the advantages that fresh pig embryo culture medium NCSU-23 and feeder layer cells are better in effect than the fresh pig embryo culture medium NCSU-23 and feeder layer cells added with 10% FBS, and the blastocyst rate of in-vitro culture of the oviduct epithelial cells as single-layer single embryos is higher and reaches 8.3%. Both pig fetal fibroblasts and cumulus cells as feeder layers show blastocysts, but are lower than oviduct epithelial cells.

Example 7 control of Single embryo feeder droplet Co-culture and conventional Multi-embryo Co-culture development Processes

Performing group culture on oocytes subjected to in vitro maturation activation: A. co-culturing in feeder layer cell microdroplets, each microdroplet containing an oocyte; B. placing into a five-hole plate according to the traditional method, covering paraffin oil for culture, and culturing in 100-150 medium/500 mu L medium; C. culturing by conventional micro method, 10-15 pieces/μ L culture medium.

TABLE 1 control of development Processes of Single embryo feeder layer droplet Co-culture and conventional Multi-embryo Co-culture

As shown in Table 1, the development of single embryo cultures was consistent with the development of the traditionally used multiple embryo cultures.

Example 8 blastocyst development Rate statistics

Obtaining the parthenogenetic blastocyst of the pig in 7 days. After the co-culture for 7d, the embryo development condition is observed under a microscope of 400X, and the blastocyst development rate is counted. See fig. 4.

Through determination and control tests, the in-vitro culture method of the porcine parthenogenetic activated embryo single embryo in the embodiment can obtain the embryo which normally develops to a blastocyst.

In summary, the feeder layer cell droplets and the target embryo are co-cultured to obtain a single embryo with normal development, the feeder layer cell droplets provided by the invention are simple to prepare, reliable raw materials can be provided for researching the development of the single embryo according to the method provided by the invention, and the single embryo culture method provided by the invention can be widely applied to the single embryo culture of various species.

Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.

Claims (1)

1. A pig single embryo in vitro culture method is characterized by comprising the following steps:

1) preparing mature oocytes in vitro of pigs:

collecting ovaries of sows; washing ovarian tissue with 75% alcohol for 1min, cleaning with sterilized and preheated normal saline for 3 times, extracting ovarian follicle with diameter of 3-8mm from ovarian surface with sterile syringe containing small amount of balanced DPBS and equipped with standard No. 20 needle, injecting the extractive solution into 50mL pointed-bottom centrifuge tube, and placing in 39 deg.C water bath; cumulus-oocyte complex (COC)_S) After precipitation, eggs were examined under a stereomicroscope after washing 2 times with DPBS; picking up COCs which are normal in shape and uniform in cytoplasm and are wrapped by at least 3 layers of granular cells, washing the detected COCs for 3 times by using DPBS (double DPBS) containing 5% fetal calf serum, then washing the COCs for 3 times by using in-vitro maturation culture solution, and transferring the COCs into a five-hole cell culture plate which contains 500 mu L of in-vitro maturation culture solution and is covered with paraffin oil; placing 100-150 COCs per drop; the liquid is pre-treated with CO₂ Balancing in an incubator for more than 2 hours; culturing in vitro at 39 deg.C for 42-44 hr with 5% CO₂Saturated humidity;

the in vitro maturation culture solution is: TCM-199+0.1% polyvinyl alcohol +3.05mM glucose +0.91mM sodium pyruvate +0.57mM L-cysteine +100IU/mL penicillin + 100. mu.g/mL streptomycin +10% (V/V) follicular fluid +10IU/mL PMSG +10IU/mL hCG;

2) parthenogenetic activation of porcine in vitro mature oocytes:

COCs matured in vitro for 42-44h were transferred into DPBS containing 0.1% hyaluronidase for digestion and gently blown with a pipette gun repeatedly to remove cumulus cells, washed 3 times with pre-warmed activation solution, placed in a fusion tank with activation solution, electrically activated with BTX-2001 fusion instrument with activation parameters: 1 direct current pulse of 30 mus and 1.1 kv/cm; the activated oocyte is washed for three times by embryo culture solution NCSU-23 preheated in advance for standby;

the formula of the activating liquid comprises: 0.25mol/L mannitol, 0.5mol/L calcium chloride, 0.5mol/L magnesium sulfate, 0.5mol/L HEPES and 0.01% (V/V) polyvinyl alcohol;

3) co-culture feeder layer cell droplet preparation:

(1) preparation of microdroplets of oviduct epithelial cell feeder layer

A. Isolation and primary culture of oviduct epithelial cells: putting a sow oviduct after obtaining an oviduct of a sow, adding sterile physiological saline added with double antibodies at 30-37 ℃ into the sterile physiological saline to be brought back to a laboratory within 2h, shearing redundant oviduct membranes by using sterile scissors, shearing the oviduct into small sections with the length of 2cm, washing for 3 times by using sterile physiological saline added with double antibodies at 35 ℃, putting the small sections into a disposable plastic dish with the diameter of 35mm, flushing epithelial cells while observing under a stereoscopic microscope, clamping a fracture on one side of the oviduct by using forceps of a clock and a watch in the operation process, sucking pancreatin with the working concentration of 0.25 percent by using a disposable syringe with the volume of 1 ml in the right hand, directly and slowly injecting from the fracture of the oviduct clamped by the forceps to flush the oviduct epithelial cells, collecting pancreatin the dish after repeating for 2-3 times, quickly adding DMEM containing 10 percent of FBS with the same volume to stop digestion, and avoiding excessive digestion, cell death, controlling the time of pancreatin in the oviduct within 5min so as to ensure the purity of the oviduct epithelial cells; centrifuging the collected liquid at 3000r/min for 5min, discarding the supernatant, adding DMEM containing 10% FBS, repeatedly centrifuging for 2 times, discarding the supernatant, and placing the obtained precipitate into a 6-well plate containing 2 ml of DMEM containing 10% FBS for culture; after culturing for 3 days, replacing a fresh culture medium, and digesting for later use when the cells grow over the bottom of the 6-pore plate;

B. feeder layer co-culture cell droplet preparation: digesting oviduct epithelial cells overgrowing with 6-well plate with 0.25% pancreatin 48h before co-culture for 2min, adding DMEM containing 10% FBS to stop digestion, gently blowing into single cell suspension with 1 ml pipette, and adjusting cell density to 10⁴The number/ml of the cells in the cell suspension is ensured to exist uniformly, the cell suspension is used for preparing the co-culture feeder layer single embryo culture microdroplet, namely, a pipettor with the measuring range of 10 mu L is used for sucking 10 mu L of cell suspension, and the cell microdroplet is made on a disposable capsule with the diameter of 35 mm; each droplet size 10. mu.L of overlay paraffin oil, placed at 39 ℃ in 5% CO₂Culturing in an incubator with saturated humidity, and culturing for 48h for cell co-culture;

alternatively, the first and second electrodes may be,

(2) cumulus granulosa cell feeder layer droplet preparation:

placing the cultured mature oocyte in a maturation solution, repeatedly blowing and beating cumulus cells except the oocyte by using a liquid transfer gun, completely blowing and picking up the oocyte, keeping the remaining cumulus granular cells in the solution, and adjusting the cell density to 10⁴The number/ml of the cells in the cell suspension is ensured to exist uniformly, the cell suspension is used for preparing the co-culture feeder layer single embryo culture microdroplet, namely, a pipettor with the measuring range of 10 mu L is used for sucking 10 mu L of cell suspension, and the cell microdroplet is made on a disposable capsule with the diameter of 35 mm; each droplet size 10. mu.L of overlay paraffin oil, placed at 39 ℃ in 5% CO₂Culturing in an incubator with saturated humidity, and culturing for 48h for cell co-culture;

4) co-culturing single embryo and feeder layer cells:

(1) co-culture of oocyte and feeder layer cell with zona pellucida attachment

Changing a cell culture medium DMEM in the microdroplets into a fresh embryo culture medium NCSU-23 2 hours before the oocytes are placed into the microdroplets for later use;

selecting in-vitro mature oocytes with first polar bodies and uniform cytoplasm obtained in the step 2), washing for 3 times in an embryo culture medium NCSU-23 balanced for 2 hours in advance, and then placing into the co-culture feeder layer single-cell microdroplets prepared in the step 3) for co-culture, wherein 1 oocyte is placed in each microdroplet; carrying out half-amount liquid change within 48 h;

alternatively, the first and second electrodes may be,

(2) co-culture of zona pellucida-removed oocytes and feeder cells

Changing a cell culture medium DMEM in the microdroplets into a fresh embryo culture medium NCSU-23 2 hours before the oocytes are placed into the microdroplets for later use;

after in vitro mature oocytes are parthenogenetically activated, selecting in vitro mature oocytes with a first polar body, uniform cytoplasm and complete cell membranes, digesting the in vitro mature oocytes with pronase with the working concentration of 0.25% to remove zona pellucida, washing the zona pellucida-free oocytes in an embryo culture medium NCSU-23 balanced for 2 hours in advance for 3 times, placing the zona pellucida-free oocytes into single embryo culture microdroplets of the co-culture feeder layer prepared in the step 3) for co-culture, placing 1 oocyte in each microdroplet, and performing half-amount liquid change for 48 hours;

5) obtaining the parthenogenetic blastocysts of the pigs in 7 days: and (4) counting the embryo cleavage rate in 36h, observing the development condition of the embryo under a microscope after co-culture for 7d, and counting the blastocyst development rate.

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