CN115354019B - In-vitro maturation culture solution for porcine oocytes and application thereof - Google Patents
In-vitro maturation culture solution for porcine oocytes and application thereof Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0608—Germ cells
- C12N5/0609—Oocytes, oogonia
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
- Y02A40/81—Aquaculture, e.g. of fish
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Abstract
The invention discloses a pig oocyte in-vitro maturation culture solution and application thereof, and belongs to the technical field of livestock breeding. The invention discloses a pig oocyte in-vitro maturation culture solution, which is prepared by adding 0.04-0.05 mg/mL or 0.14-0.18 mg/mL of citric acid into a basic culture solution. The invention can effectively obtain a large number of high-quality oocytes, provides support for the technologies of in vitro fertilization, parthenogenesis activation, nuclear transfer preparation of cloned embryos and the like, and plays an important role in the aspects of propagation of improved livestock, animal model construction, transgenic animal production and the like.
Description
Technical Field
The invention relates to the technical field of livestock breeding, in particular to a pig oocyte in-vitro maturation culture solution and application thereof.
Background
In vitro maturation culture of porcine oocytes is an important link in livestock breeding technology. Mature oocytes are widely applied to the technical fields of in-vitro fertilization, parthenogenesis, nuclear transfer, preparation of cloned embryos and the like, and further can provide support for propagation of improved livestock, animal model construction, transgenic animal production and the like. However, the problems of inconsistent maturation rate, inconsistent development quality and the like exist in the in-vitro maturation process of the pig oocyte at present, and the subsequent basic research and the practical use of production are restricted, so that the in-vitro maturation of the pig oocyte is improved. The reason for the low in-vitro maturation efficiency of oocytes is that compared with in-vivo maturation, the in-vitro maturation culture system cannot completely reduce the in-vivo microenvironment due to the difference of the in-vivo environment of the mother body and numerous other influencing factors, so that the establishment of an in-vitro culture system with higher stability and higher oocyte maturation efficiency is urgently needed.
At present, it is not clear what core factors beneficial to oocyte development are added in the in vitro maturation process, and factors which are widely considered to contribute to oocyte maturation include hormones, growth factors, cytokines and other components.
Therefore, providing a swine oocyte in vitro maturation culture solution and application thereof is a problem to be solved by those skilled in the art.
Disclosure of Invention
In view of the above, the invention provides a swine oocyte in vitro maturation culture solution and application thereof.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
the pig oocyte in-vitro maturation culture solution is characterized in that 0.04-0.05 mg/mL or 0.14-0.18 mg/mL of citric acid is added into the basic culture solution.
Further, the pig oocyte in-vitro maturation culture solution is added with 0.048mg/mL or 0.144mg/mL of citric acid in the basal culture solution.
Further, the basal culture broth is based on M199 culture broth without HEPES, 90% volume fraction TCM199, 10% volume fraction pig follicular fluid are added, and 1mg/mL L-cysteine, 0.44mg/mL sodium pyruvate, 10ng/mL epidermal growth factor, 50ng/mL insulin, 5IU/mL gonadotropin, 5IU/mL chorionic gonadotropin are added.
Further, the application of the in-vitro maturation culture solution of the porcine oocytes in improving the in-vitro maturation rate and the development quality of the porcine oocytes.
The early stage of subject group screening shows that the citric acid level in the in vivo follicular fluid is positively correlated with the oocyte development potential, and simultaneously, the citric acid is also proved to be used as an initial energy substrate in tricarboxylic acid circulation, thereby being beneficial to the maturation development process of the oocyte. The tricarboxylic acid cycle is a ubiquitous metabolic pathway in aerobic organisms, regulates the metabolic growth and development of organisms, and citric acid promotes Ca by enhancing the tricarboxylic acid cycle metabolic pathway, increasing ATP levels 2+ The oscillation is helpful to improve the activation of the oocyte, further improve the in vitro maturation of the oocyte, and promote the activation of the oocyte and the subsequent embryo initiationPlays an irreplaceable role in the middle-jiao.
The application of the citric acid in the porcine oocyte basic culture solution, in particular to the application of the citric acid in improving the maturation rate and the development quality of the porcine oocyte, namely, the application of the citric acid in the basic culture solution can effectively improve the in-vitro maturation rate and the development quality of the porcine oocyte, ensure the mitochondrial activity of the porcine oocyte, improve the normal proportion of the spindle body and chromosome of the oocyte, and improve the number of blastula cells, thereby obtaining a large number of high-quality in-vitro mature oocytes and providing support for basic scientific research and production practice.
Compared with the prior art, the invention discloses the in-vitro maturation culture solution of the porcine oocyte and the application thereof, and the utilization rate of the small follicular oocyte can be greatly improved by adding 0.048mg/mL citric acid into the basic culture solution, the maturation rate is improved by approximately 20%, the normal proportion of the spindle body is improved by approximately 30%, and the number of blastula cells is improved by approximately 25%. And the maturation rate of oocytes is improved by 15% by adopting 0.144mg/mL of citric acid, and the number of blastula cells is improved by approximately 35%. The proposal can effectively obtain a large amount of high-quality oocytes, provides support for the technologies of in vitro fertilization, parthenogenesis activation, nuclear transfer, clone embryo preparation and the like, and plays an important role in the aspects of propagation of improved livestock, animal model construction, transgenic animal production and the like.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments or the description of the prior art will be briefly described below, and it is obvious that the drawings in the following description are only embodiments of the present invention, and that other drawings can be obtained according to the provided drawings without inventive effort for a person skilled in the art.
FIG. 1 is a diagram showing the degree of expansion of a cumulus in an in vitro maturation process of an oocyte according to the present invention with or without the addition of citric acid to a basal medium; the control group is indicated by arrows that the expansion degree of the cumulus cells is low, and the treatment group (the citric acid concentration is 0.048 mg/mL) is obviously increased; each group of two pictures is displayed with a scale of 200 μm;
FIG. 2 is a diagram showing the morphology of the first polar row and periova in the MII period of the oocyte added (citric acid concentration is 0.048 mg/mL) or not added in the basic culture solution in the in-vitro maturation process of the oocyte of the present invention; two pictures in each group are used for displaying; the arrow indicates a first electrode body, and the scale is 50 μm;
FIG. 3 is a graph showing mitochondrial activity after maturation of oocytes with or without addition of citrate to a basal medium during in vitro maturation of oocytes in accordance with the present invention;
wherein A is the mitochondrial activity fluorescence expression condition of oocytes in the MII stage of a control group and a treatment group (0.048 mg/mL of citric acid is added into a basic culture solution), and a confocal microscope is used for photographing; two pictures in each group are used for displaying; the scale bar is 50 mu m; b is the quantitative level of mitochondrial activity fluorescence of oocytes in the MII phase of a control group (Ctrl) and a treatment group (0.048 mg/mL and 0.144mg/mL of citric acid are added into a basic culture solution), and the quantitative level is analyzed by using software Image J;
FIG. 4 is a diagram showing the status of spindle and chromosome after maturation of oocytes with or without addition of citric acid to the basal medium during in vitro maturation of oocytes in accordance with the present invention;
wherein, A is the shape of the spindle of the oocyte in the MII stage of the control group and the treatment group (0.048 mg/mL citric acid is added into the basic culture solution), and a confocal microscope is used for photographing; two pictures in each group are used for displaying; the scale bar is 50 mu m; b is a control group (Ctrl) and a treatment group (0.048 mg/mL and 0.144mg/mL of citric acid are added in basic culture solution), the normal proportion percentage of the spindle shape of the oocyte in the MII stage is set, three groups of repetition are set for control and treatment, 15-20 oocytes in the MII stage are detected in each group of repetition, and finally, the result difference statistics is analyzed by using SPSS statistical software;
FIG. 5 is a graph showing the number of blasts after maturation of oocytes with or without the addition of citric acid to the basal medium during in vitro maturation of oocytes in accordance with the present invention.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
HEPES-free M199 culture solution is a product of Semerle Feichi technologies, and other reagents without special instructions are all sigma Aldrich products. Embryo water is a solvent for reagents required for embryo culture configuration, and is derived from sigma, W1503-500ML.
The energy required by the embryo is provided by the mitochondria of the ovum in the process from fertilization to embryo implantation, and the quality of the ovum is poor and the chromosome of the embryo is abnormal along with the growth of the animal, the quantity of the mitochondria is gradually reduced and the function is continuously weakened, so that when the oocyte of the target pig is cultivated and matured in vitro, the age difference of individual sows from the ovary is obvious, the individuals are different, the in vitro maturing rate of the oocyte is low and the development is inconsistent, and the citric acid (tricarboxylic acid) is circulated to serve as a final metabolic pathway of three nutrients, thereby playing an important role in promoting the maturation of the oocyte and guaranteeing the development potential development of the oocyte. Therefore, the invention achieves the aim of improving the in vitro maturation rate and the development quality of the porcine oocyte by adding citric acid.
Example 1
(1) Physiological saline
Into a 600mL glass bottle was added 500mL deionized water, 4.5g sodium chloride was added, and the mixture was stirred until completely dissolved and autoclaved. The sterilized normal saline is preserved at normal temperature, and is placed in a constant temperature box at 38 ℃ for overnight preheating before use.
(2) Pig follicular fluid
Collecting fresh ovary of pig, placing in a physiological saline thermos bottle at 38deg.C, extracting follicular fluid of ovarian surface follicle with 12G needle syringe, selecting follicular diameter of 2-5 mm (small follicle), collecting follicular fluid in 50mL centrifuge tube, standing in 38 deg.C incubator for 30min, collecting upper follicular fluid, centrifuging at 3000rpm for 30min, collecting lower layer of cumulus granulosa cell-oocyte complex (COCs), filtering the supernatant with 0.22 μm filter, and storing at-20deg.C.
(3) In vitro maturation culture solution for porcine oocytes
Control group: in vitro maturation broth without citric acid.
Based on M199 without HEPES, 90% volume fraction TCM199, 10% volume fraction swine follicular fluid, 1mg/mL L-cysteine, 0.44mg/mL sodium pyruvate, 10ng/mL epidermal growth factor, 50ng/mL insulin, 5IU/mL gonadotropin, 5IU/mL chorionic gonadotropin were added.
Treatment group: in vitro maturation culture solution containing citric acid with different concentrations
Based on M199 without HEPES, 90% volume fraction TCM199, 10% volume fraction pig follicular fluid, 1mg/mL L-cysteine, 0.44mg/mL sodium pyruvate, 10ng/mL epidermal growth factor, 50ng/mL insulin, 5IU/mL gonadotropin, 5IU/mL chorionic gonadotropin, citric acid at different concentrations (0.048 mg/mL, 0.096mg/mL, 0.144mg/mL, 0.192mg/mL, 0.48 mg/mL) were added.
Experiments show that the addition of 0.048mg/mL or 0.144mg/mL of citric acid into the basic culture solution is good, and the treatment group in the subsequent experiments is added at 0.048mg/mL or 0.144 mg/mL.
(4) TLH (Tyrode's medium) -PVA pig oocyte operating fluid
6.663g of sodium chloride, 0.237g of potassium chloride, 0.168g of sodium bicarbonate, 0.041g of sodium dihydrogen phosphate, 1.868mL of sodium lactate, 0.102g of magnesium chloride hexahydrate, 0.294g of calcium chloride dihydrate, 2.383g of HEPES, 0.022g of sodium pyruvate, 0.025g of gentamicin, 0.065g of penicillin, 0.100g of polyvinyl alcohol and 0.010g of phenol red are added into ultrapure water, and the mixture is fully dissolved, fixed to a volume of 1L, and the pH value is adjusted to 7.2-7.4, and the osmotic pressure is 295-300 mOsm.
1) In vitro maturation culture of porcine oocytes
Collecting fresh ovaries of pigs, placing the fresh ovaries in a physiological saline vacuum bottle at 38 ℃, extracting follicular fluid of follicles on the surfaces of the ovaries by using a 12G needle head injector, selecting small follicles with the follicular diameter of 2-4 mm, collecting the extracted follicular fluid in a 50mL centrifuge tube, standing for 30min in a 38 ℃ constant temperature box, taking follicular fluid as the upper layer and cumulus granulosa cell-oocyte complexes (COCs) as the lower layer, pouring the follicular fluid as the upper layer, leaving the COCs as the lower layer, adding an equal volume of TLH-PVA pig oocyte operating fluid, reversing and uniformly mixing, pouring the mixture into a 100mm culture dish, selecting under a split microscope, selecting three or more layers of COCs of cumulus cells by using a 500 mu m glass needle, washing three times in vitro maturation culture fluid without citric acid, and transferring the three times into a control or treatment group for maturation culture.
Control group: adding in vitro maturation culture solution of pig oocyte without citric acid into 96-well plate, adding 150 μl of culture solution into each well, coating paraffin oil 50 μl, and heating at 385 deg.C and 5% CO 2 Preheating for more than 5 hours in a saturated humidity incubator, and placing COCs in small follicles after selection and washing in mature holes, wherein 30 COCs are placed in each hole.
Treatment group 1: adding 0.048mg/mL pig oocyte in vitro maturation culture solution containing citric acid into 96-well plate, adding 150 μl of culture solution into each well, coating paraffin oil 50 μl, and heating at 38.5deg.C and 5% CO 2 Preheating for more than 5 hours in a saturated humidity incubator, and placing selected and washed small follicle COCs in mature holes, wherein each hole is 30.
Treatment group 2: adding 0.144mg/mL of citric acid-containing pig oocyte in vitro maturation culture solution into 96-well plates, adding 150 μl of culture solution into each well, coating paraffin oil 50 μl, and heating at 38.5deg.C and 5% CO 2 Preheating for more than 5 hours in a saturated humidity incubator, and placing selected and washed small follicle COCs in mature holes, wherein each hole is 30.
2) MII phase oocyte collection
Mature COCs were pipetted into a 0.5mL centrifuge tube using a 200 μl pipette and mixed with hyaluronidase solution (0.25 g hyaluronidase+100 mL embryo water, hyaluronidase accession number sigma H3506): COCs and maturation fluids = 1:50 volumes of hyaluronidase solution was added, and the mixture was shaken for 5min with a shaker, and centrifuged at 1000rpm for 1min. COCs at the bottom of the centrifuge tube are sucked by a glass needle with the caliber of 200 mu m, washed three times in TLH-PVA solution, and cumulus granulosa cells are fully removed. And then picking out the oocytes of the first polar body by using a 200 mu m glass needle under a stereoscopic microscope, namely the mature MII-stage oocytes. The above method was applicable in both the treatment group and the control group.
Maturation rate = number of oocytes discharged from the first polar body/total number of oocytes
TABLE 1 efficiency statistics of mature oocytes obtained by adding different concentrations of citric acid to basal broths
Note that: at least 3 experiments per group; the maturation rate is shown in mean ± standard deviation; the data in the same column are marked with different letters to indicate significant differences (p <0.05, one-way anova Duncan's test).
FIG. 1 shows the degree of expansion of a cumulus in a basal medium with or without citric acid added during in vitro maturation of oocytes; FIG. 2 shows the morphology of the first polar in vitro row and peri-oval gap during MII phase of oocytes with or without citric acid added to the basal medium during in vitro maturation of the oocytes.
Test results: as is clear from table 1, fig. 1 and fig. 2, when 0.048mg/mL and 0.144mg/mL of citric acid were added to the culture solution, the maturation rate was significantly improved, the expansion degree of the cumulus cells was more sufficient, the polar body discharge was more remarkable, and the treatment groups were added at 0.048mg/mL and 0.144mg/mL in the subsequent experiments, as compared with the control group without citric acid.
TABLE 2 efficiency statistics of mature oocytes obtained with or without addition of citric acid to basal medium
Note that: at least 3 experiments per group; the maturation rate is shown in mean ± standard deviation; the data in the same column are marked with different letters to indicate significant differences (p <0.05, one-way anova Duncan's test).
As shown in Table 2, the maturation rate was increased by about 20% after 0.048mg/mL of citric acid was added to the treatment group 1 pig oocyte basal medium, and the maturation rate was increased by about 15% after 0.144mg/mL of citric acid was added to the treatment group 2 pig oocyte basal medium, as compared with the control group.
3) Pig oocyte mitochondrial activity detection
Pig mature oocyte mitochondrial activity assay live staining was performed using MitoTracker Red CMXRos (mitochondrial activity fluorescent probe, bi yun). Taking control group and treated group MII oocytes, respectively placing in basal culture solution containing 200nM MitoTracker Red CMXRos, in dark environment at 37deg.C and 5% CO 2 After 30min incubation, the oocytes are washed three times in basic culture solution, and finally the oocytes are placed on a glass slide for tabletting, and a laser scanning confocal microscope is used for observing and counting mitochondrial activity. The results are shown in FIG. 3.
As can be seen from FIG. 3, when 0.048mg/mL or 0.144mg/mL of citric acid was added to the porcine oocyte basal medium, there was no significant difference in mitochondrial activity, but the 0.048mg/mL treated group had a more uniform level of mitochondrial activity, indicating that oocyte development was more consistent.
4) Pig oocyte spindle body and chromosome morphology detection
Pig mature oocyte spindle and chromosome morphology detection were stained with anti-alpha-tubuLin (Biyun) and control and treatment groups of MII oocytes were allowed to stand in fixative solution for 30min at room temperature, washed three times with wash solution and transferred into permeabilization solution for 30min at room temperature. After three times of washing, the mixture was transferred into a blocking solution and allowed to stand at room temperature for 2 hours, and then the control group and the treated group MII oocytes after standing at room temperature were respectively placed in a basal culture solution containing anti-alpha-tubuLin (1:200) in a dark environment at 37 ℃ and 5% CO 2 After 30min incubation, the oocytes were washed three times in basal medium, transferred to DAPI and visualized by tabletting. The results are shown in FIG. 4.
As can be seen from FIG. 4, compared with the control group (20.00.+ -. 10.00%), when 0.048mg/mL or 0.144mg/mL of citric acid was added to the pig oocyte basal medium, the spindle integrity ratio was significantly increased (53.33.+ -. 5.77%; 30.74.+ -. 8.91%), and the effect of the 0.048mg/mL treatment group was more significant, and the spindle normal ratio was increased by about 30%.
5) Detection of pig in vitro fertilization blastula rate and blastula cell number
Collecting in vitro fertilized blasts developed from a control group and a treatment group until the 6 th day, and marking the nuclei of the blasts by immunofluorescence staining technique, wherein the specific method is as follows: three groups of blastula are respectively placed in a fixing solution for 30min at room temperature, washed three times by a washing solution, transferred into a permeabilization solution and placed for 30min at room temperature. After washing three times again, the blasts were transferred into a blocking solution and allowed to stand at room temperature for 2 hours, after which the blasts were placed on a slide glass on which a DAPI (nuclear stain) containing blocking solution was dropped for tabletting, and the number of blasts (DAPI-positive stained nuclei number) was counted under a fluorescence microscope. The results are shown in Table 3 and FIG. 5.
TABLE 3 efficiency and cell count statistics for obtaining in vitro fertilized blasts using mature oocytes of the treatment and control groups
Note that: each set of experiments was repeated three times; blastocyst rate and cell number are shown as mean ± standard deviation; the data in the same column are marked with different letters to indicate significant differences (p <0.05, one-way anova Duncan's test).
As can be seen from Table 3 and FIG. 5, compared with the control group, when 0.048mg/mL (treatment group 1) or 0.144mg/mL (treatment group 2) of citric acid is added into the pig oocyte basic culture solution, the development efficiency of the pig in-vitro fertilized embryo into the blastocyst can be remarkably promoted, the efficiency of the pig in-vitro fertilized embryo is improved by about 10%, and meanwhile, the cell number of the in-vitro fertilized blastocyst is remarkably improved, so that the improvement of the whole development quality of the blastocyst is shown.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (2)
1. The application of the pig oocyte in-vitro maturation culture solution in improving the in-vitro maturation rate and the development quality of the pig oocyte is characterized in that 0.04-0.05 mg/mL or 0.14-0.18 mg/mL of citric acid is added into a basic culture solution; the basal culture solution is based on M199 culture solution without HEPES, 90% volume fraction TCM199 and 10% volume fraction pig follicular fluid are added, and 1mg/mL L-cysteine, 0.44mg/mL sodium pyruvate, 10ng/mL epidermal growth factor, 50ng/mL insulin, 5IU/mL gonadotropin and 5IU/mL chorionic gonadotrophin are added in a supplementing manner.
2. The use of the in vitro maturation culture solution for porcine oocytes according to claim 1 for improving the in vitro maturation rate and the development quality of porcine oocytes, wherein 0.048/mg/mL or 0.144/mg/mL of citric acid is added to the basic culture solution.
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| CN102482644A (en) * | 2009-05-14 | 2012-05-30 | 阿德莱德研究和创新私人有限公司 | Methods for the collection and maturation of oocytes |
| CN110951678A (en) * | 2019-12-26 | 2020-04-03 | 华中农业大学 | Culture solution for promoting in-vitro maturation of porcine oocytes |
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| CN102482644A (en) * | 2009-05-14 | 2012-05-30 | 阿德莱德研究和创新私人有限公司 | Methods for the collection and maturation of oocytes |
| CN110951678A (en) * | 2019-12-26 | 2020-04-03 | 华中农业大学 | Culture solution for promoting in-vitro maturation of porcine oocytes |
Non-Patent Citations (2)
| Title |
|---|
| Omnia ELGENDY等.5-Aminolevulinic acid combined with sodium ferrous citrate mitigates effects of heat stress on bovine oocyte developmental competence.Journal of Reproduction and Development.2022,第68卷(第4期),摘要. * |
| 梁力丰.重金属耦合剂和抗氧化剂在小鼠卵母细胞体外成熟前老化中的保护作用.CNKI博士电子期刊.2017,第3.4-3.6节，第65-68页. * |
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