Name: Mouse Oocyte In Vitro Maturation, Fertilization, and Culture of Preimplantation Embryos (Video) | JoVE Journal
Uploaded: 2026-05-29T15:15:00.000+00:00
Duration: 9 min 24 s
Description: Learn mouse oocyte in vitro maturation, fertilization, and preimplantation embryo culture to blastocyst stage. These integrated protocols enable controlled studies of mammalian reproductive and developmental biology.

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5 minutes

We thank Professor Yi-Liang Miao (Huazhong Agricultural University) for valuable advice and guidance in the optimization of this protocol. This work was supported by the Shaanxi Provincial Natural Science Basic Research Program (2025JC-XKZX-03).

All animal experiments were approved by the Institutional Animal Care and Use Committee of Northwest A&#38;F University, China (permit number: 20230503).
1. Animal maintenance
<ol>
	<li>Procure wild-type female C57BL/6 and male DBA/2 mice and keep them in specific pathogen-free (SPF) conditions, with a 12 h light-dark cycle at a temperature of 20&#8211;22 &#176;C and 55% &#177; 10% humidity. Provide a breeding diet and water ad libitum. House male mice singly (one male per cage).</li>
</ol>
2. Preparation of media 
<ol>
	<li>To prepare a 50 IU/mL PMSG working solution, dissolve 5,000 IU pregnant mare serum gonadotropin (PMSG) in 100 mL of sterile phosphate-buffered saline (PBS). 
	NOTE: Aliquot the solution into 1 mL single-use portions and store them at &#8211;20 &#176;C. Thaw each aliquot at room temperature immediately before use.</li>
	<li>To prepare IVM culture medium, add the ingredients listed in Table 1 to 100 mL of Medium 199 (M199) base medium. Adjust the pH to 7.3&#8211;7.4, then sterile-filter the medium through a 0.22 &#181;m filter into sterile medium bottles. Label the bottle &#8220;IVM&#8221; with initials, date made, and expiration date, and store at 4 &#176;C for up to 1 month.</li>
	<li>To prepare a 0.1% (w/v) hyaluronidase solution, dissolve 10 mg hyaluronidase in 10 mL of M2 medium. 
	NOTE: Store in single-use aliquots of 1 mL at &#8211;20 &#176;C and warm to 37 &#176;C before use.</li>
</ol>
3. Preparation of dishes (Figure 2)
NOTE: Adjust the number of IVM, IVF, and IVC drops according to the expected numbers of COCs and embryos, using 20&#8211;30 COCs per 100 &#181;L drop for IVM and IVF and 20&#8211;30 embryos per 100 &#181;L drop for embryo culture.
<ol>
	<li>Prepare an appropriate number of 35 mm oocyte collection dishes, each containing 2 mL of M2 medium supplemented with 0.2 mM 3-isobutyl-1-methylxanthine (IBMX). Use one dish for ovary puncture and COC collection (one dish per two females), and another for washing COCs. Prewarm the dishes to 37 &#176;C before use.</li>
	<li>Prepare IVM dishes.
	<ol>
		<li>In a 60 mm dish, prepare five 100 &#181;L drops of IVM medium for washing (label the dish as &#8220;IVM wash&#8221;).</li>
		<li>In a separate 60 mm dish, prepare multiple 100 &#181;L drops of IVM medium for maturation (label the dish as &#8220;IVM&#8221;).</li>
		<li>Carefully add sufficient mineral oil to cover the drops, and equilibrate the dishes at 37 &#176;C in a 5% CO2 incubator for at least 2 h before starting oocyte collection.</li>
	</ol>
	</li>
	<li>Prepare IVF dishes.
	<ol>
		<li>In a 60 mm dish, prepare one or two 300 &#181;L drops of preequilibrated embryo culture medium for sperm release and capacitation (label the dish as &#8220;Sperm capacitation&#8221;). 
		NOTE: The second capacitation drop is prepared as a reserve for cases in which sperm motility in the first drop is insufficient, a new epididymal sperm sample must be collected and capacitated, or sperm concentration needs to be adjusted before insemination.</li>
		<li>Prepare two separate 60 mm dishes, each containing five 100 &#181;L drops of preequilibrated culture medium for washing (label the dish as &#8220;IVF wash&#8221;). 
		NOTE: Use one &#8220;IVF wash&#8221; dish to wash IVM-matured COCs before insemination, and use the other &#8220;IVF wash&#8221; dish to wash presumptive zygotes after co-incubation to remove cumulus cells and residual sperm.</li>
		<li>In a separate 60 mm dish, prepare multiple 100 &#181;L drops of preequilibrated embryo culture medium for insemination (label the dish as &#8220;IVF&#8221;). 
		NOTE: Preequilibrated embryo culture medium is used for both sperm capacitation and insemination to maintain consistent conditions during sperm preparation and fertilization.</li>
		<li>Carefully add sufficient mineral oil to cover the drops, and equilibrate the dishes at 37 &#176;C in a 5% CO2 incubator for at least 12 h before starting the sperm capacitation procedure.</li>
	</ol>
	</li>
	<li>Prepare IVC dishes.
	<ol>
		<li>In a 60 mm dish, prepare five 100 &#181;L drops of preequilibrated embryo culture medium for washing embryos (label the dish as &#8220;IVC wash&#8221;).</li>
		<li>In a separate 60 mm dish, prepare multiple 100 &#181;L drops of preequilibrated embryo culture medium for embryo culture&#160;(label the dish as &#8220;IVC&#8221;).</li>
		<li>Carefully add sufficient mineral oil to cover the drops, and equilibrate the dishes at 37 &#176;C in a 5% CO2 incubator for at least 12 h before starting embryo culture.</li>
	</ol>
	</li>
</ol>
4. Ovary collection
<ol>
	<li>Administer 10 IU PMSG intraperitoneally to 8&#8211;10-week-old female C57BL/6 mice.</li>
	<li>At 48 h after PMSG injection, euthanize the mouse by cervical dislocation and position it supine. Spray the abdomen with 70% ethanol to wet the fur and prevent it from entering the abdominal cavity.</li>
	<li>Using fine dissecting scissors, make a small lower abdominal skin incision and gently open the skin with gloved hands to expose the peritoneal membrane.</li>
	<li>Open the peritoneal membrane and gently move the intestines aside. Locate the uterine horn and follow it toward the ovary. Identify the ovary near the kidney, typically enclosed by a fat pad.</li>
	<li>Use fine dissecting scissors to separate the ovary from the uterine horn, and trim away excess connective tissue and fat.</li>
	<li>Immediately transfer the ovaries to pre-warmed M2 medium supplemented with IBMX. Repeat the procedure to collect the contralateral ovary and transfer it to the same dish.</li>
	<li>Place the dish under a stereomicroscope and proceed with follicle puncture and COCs collection.</li>
</ol>
5. COCs collection and culture
<ol>
	<li>Under a stereomicroscope, gently puncture visible antral follicles with a sterile syringe needle to release COCs in the M2 medium. 
	NOTE: When collecting mouse GV-stage oocytes, preferentially puncture visibly large antral follicles on the ovarian surface, which typically appear bulging and semi-translucent with a clear antral cavity.</li>
	<li>Using a finely pulled glass Pasteur pipette, collect the released COCs and transfer them to a clean 35-mm dish containing pre-warmed M2 medium supplemented with IBMX.</li>
	<li>Wash the COCs by serial transfer to a clean area of the 35 mm dish as needed to remove detached granulosa cells and tissue debris, leaving debris behind at each transfer (Figure 3). 
	NOTE: Use only COCs with three or more compact cumulus-cell layers and uniformly granulated cytoplasm without clear spaces for the subsequent steps9,10. Denuded GV oocytes and partially cumulus-enclosed GV oocytes are excluded from this protocol. Under our experimental conditions, an average of 26 immature COCs was obtained per female mouse.</li>
	<li>After debris removal, transfer the COCs to the IVM wash dish and wash them by sequential transfer through five 100 &#181;L drops of IVM wash medium. 
	NOTE: During transfers, minimize carryover volume to prevent cross-contamination between drops.</li>
	<li>Transfer the washed COCs into 100 &#181;L drops of IVM medium (20&#8211;30 COCs per drop) and culture at 37 &#176;C in 5% CO2 for 14 h. 
	NOTE: Collect, wash, and transfer COCs to IVM medium with minimal delay to help maintain developmental potential.</li>
	<li>(Optional) After IVM, briefly incubate the COCs in 1 mg/mL hyaluronidase and gently pipette them repeatedly with a micropipette until the cumulus cells are completely removed. 
	NOTE: Limit hyaluronidase exposure to no more than 3 min, as prolonged exposure may damage the oocytes.</li>
	<li>(Optional) Wash the denuded oocytes in fresh M2 medium on a heated stage, then assess oocyte maturation under a stereomicroscope.</li>
	<li>(Optional) Oocytes with an extruded first polar body are considered mature and at the metaphase II (MII) stage. Calculate the maturation rate as the number of oocytes with an extruded first polar body divided by the total number of oocytes cultured. 
	NOTE: Steps 5.6&#8211;5.8 are used to assess oocyte maturation after IVM by denuding a designated assessment cohort before PB1 scoring. These steps are not required for COCs that proceed directly to IVF.</li>
</ol>
6. Sperm collection and preparation
NOTE: This section describes the collection and capacitation of fresh sperm. Prior to insemination, freshly released sperm should be incubated in preequilibrated embryo culture medium at 37 &#176;C for 1 h to allow capacitation.
<ol>
	<li>Euthanize one mature male DBA/2 mouse (10&#8211;16 weeks of age) and remove its cauda epididymides, avoiding as much fat, blood, and tissue fluid as possible (Figure 4).</li>
	<li>For each male, transfer both cauda epididymides into a 300 &#181;L drop of preequilibrated G-IVF medium for sperm release and capacitation.</li>
	<li>Immobilize the cauda epididymis using a pair of forceps, make five to seven cuts in the cauda epididymis with a sterile syringe needle, and gently squeeze the tissue to release concentrated sperm. 
	NOTE: Evaluate sperm motility and proceed only when the majority of sperm show progressive motility (PR), defined as active forward movement, rather than non-progressive motility (NP) or immotility11.</li>
	<li>Incubate the dish at 37 &#176;C in 5% CO&#8322; for 1 h to allow sperm capacitation.</li>
</ol>
7. In vitro fertilization
<ol>
	<li>After 14 h of IVM, wash the COCs by sequential transfer through five 100 &#181;L drops of IVF wash medium, and then transfer the washed COCs into the IVF insemination drops (20&#8211;30 COCs per 100 &#181;L drop).</li>
	<li>Preincubate the fertilization dish containing COCs at 37 &#176;C in 5% CO2 for 30 min before insemination.</li>
	<li>After capacitation, determine sperm concentration using a hemocytometer.</li>
	<li>Based on the sperm concentration, calculate the required inoculum volume and add capacitated sperm to each IVF drop to achieve a final concentration of 4 &#215; 105 &#8211;1 &#215; 106 sperm/mL. 
	NOTE: After sperm addition, confirm that motile sperm are present in each insemination drop.</li>
	<li>Incubate the IVF dishes containing COCs and sperm at 37 &#176;C in a 5% CO&#8322; incubator for 4&#8211;6 h.</li>
</ol>
8. Embryo culture
<ol>
	<li>After 4&#8211;6 h of incubation, use a finely pulled glass Pasteur pipette with an inner diameter slightly larger than the oocyte to aspirate presumptive zygotes in minimal volume and transfer them into the IVF wash drops.</li>
	<li>Wash the presumptive zygotes by sequential transfer through five IVF wash drops, gently pipetting within each drop to remove excess sperm and debris (Figure 5).</li>
	<li>Transfer the washed presumptive zygotes to the IVC wash dish and wash them by sequential transfer through five prewarmed embryo culture medium wash drops, minimizing carryover of IVF medium.</li>
	<li>Place the presumptive zygotes into embryo culture medium culture drops and culture at 37 &#176;C in 5% CO&#8322; (20&#8211;30 embryos per 100 &#181;L drop).</li>
	<li>At 6&#8211;8 h after insemination, assess fertilization under a stereomicroscope by scoring the presence of male and female pronuclei, and discard oocytes that retain only one polar body and show no visible pronuclei.</li>
	<li>At 24 h after insemination, count the number of two-cell embryos and calculate the two-cell rate as (two-cell embryos / inseminated oocytes).</li>
	<li>After counting, remove and discard the uncleaved embryos, and continue culturing the remaining cleaved embryos. 
	NOTE: At this stage, embryos may be vitrified, transferred to recipient females, or maintained in culture until the blastocyst stage.</li>
	<li>Continue to culture the embryos under the same incubation conditions until 96 hours post-insemination (hpi). Assess blastocyst formation at 96 hpi based on morphological criteria. 
	NOTE: No further medium change is required during this period. A normal blastocyst is identified by the presence of a visible blastocoel cavity, a distinct inner cell mass, and an intact trophectoderm layer.</li>
</ol>

The authors have no conflicts of interest to disclose.

This protocol provides a standardized procedure for mouse oocyte in vitro maturation, in vitro fertilization, and in vitro culture using immature COCs, enabling reproducible generation and evaluation of preimplantation embryos through the blastocyst stage under defined in vitro conditions. In many studies, IVM outcomes are sensitive to laboratory-specific differences in media, incubation times, and handling, which can reduce developmental competence after IVM12<...

Understanding the oocyte quality and developmental competence is essential for successful fertilization and subsequent embryonic development1,2. Oocyte maturation requires not only accurate chromosome segregation during nuclear maturation, but also cytoplasmic maturation, including organelle redistribution and the accumulation of maternal mRNAs and proteins3. Early embryonic development relies on maternally provided mRNAs and proteins, and the maternal stores in the oocyte sustain preimplantation development. However, the identities and functions of many maternal regulators of mammalian embryogenesis remain incompletely understood4. Therefore, further systematic studies are needed to elucidate the mechanisms underlying oocyte maturation, fertilization, and embryo development.
The mouse is a widely used model system for investigating preimplantation embryo development. In vitro maturation (IVM) refers to the collection of immature oocytes from the ovary and their maturation under controlled laboratory conditions5. Subsequently, in vitro fertilization (IVF) enables mature oocytes to be co-incubated with capacitated sperm to generate zygotes for early embryonic development studies. However, relative to conventional IVF using in vivo&#8211;matured oocytes, IVM-derived oocytes often exhibit lower maturation and fertilization rates and reduced developmental competence6. These limitations are largely attributed to asynchrony between nuclear and cytoplasmic maturation under in vitro culture conditions. Moreover, meiotic spindle stability in mouse oocytes is sensitive to changes in pH and temperature during IVM, and spindle disturbance may contribute to maturation arrest7. Consequently, the reduced competence of IVM-derived oocytes remains a major barrier, limiting efficient embryo production for developmental and mechanistic studies.
Mouse preimplantation embryos can also be obtained by collecting in vivo&#8211;derived zygotes and culturing them in vitro (IVC). In vivo&#8211;derived zygotes collected from the oviducts provide a relatively synchronized starting point for experiments focused on post-fertilization events8. However, oocyte maturation and the accumulation of maternal factors occur prior to fertilization in vivo; thus, in vivo&#8211;derived zygotes are less amenable to experimental manipulation of maturation conditions or maternal stores to assess their effects on subsequent embryo development4. Therefore, optimizing IVM and IVF protocols and adopting standardized operating procedures with rigorous quality control (e.g., strict management of temperature, pH, and handling practices) are essential to minimize procedure-related variability and protect oocyte and embryo developmental competence.
Here, we describe an IVM&#8211;IVF&#8211;IVC protocol detailing media preparation, COCs isolation and selection, IVM and IVF procedures, and in vitro embryo culture (Figure 1). The protocol incorporates practical, stage-specific checkpoints for routine quality control, including first polar body (PB1) extrusion, formation of male and female pronuclei (2PN), two-cell cleavage, and blastocyst formation. This protocol enables reproducible and efficient generation of preimplantation embryos under defined conditions, supporting mechanistic studies, culture optimization, and downstream applications in developmental and reproductive biology.

<table><tbody><tr><td>35 mm Petri dish</td><td>Thermo Fisher Scientific</td><td>150255</td><td></td></tr><tr><td>60 mm Petri dish</td><td>Thermo Fisher Scientific</td><td>150270</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma-Aldrich</td><td>A8806</td><td></td></tr><tr><td>Breeding diet (rodent breeder chow)</td><td>Keao Xieli (Beijing ,China)</td><td>N/A</td><td></td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; incubator</td><td>Thermo Fisher Scientific</td><td>HERAcell 150i</td><td></td></tr><tr><td>D-glucose</td><td>Sigma-Aldrich</td><td>G7021</td><td></td></tr><tr><td>Disposable Pasteur Pipet</td><td>Kimble</td><td>DWK63A53WT-1000EA</td><td></td></tr><tr><td>Epidermal growth factor</td><td>Gibco</td><td>PHG0313</td><td></td></tr><tr><td>Fine dissecting scissors</td><td>Sigma-Aldrich</td><td>S3146-1EA</td><td></td></tr><tr><td>Fine-tip forceps</td><td>Rhino</td><td>SW14</td><td></td></tr><tr><td>Follicle-stimulating hormone</td><td>Millipore</td><td>869001</td><td></td></tr><tr><td>G-1 PLUS</td><td>Vitrolife</td><td>10128</td><td></td></tr><tr><td>G-IVF PLUS</td><td>Vitrolife</td><td>10136</td><td>(preequilibriated) embryo culture medium</td></tr><tr><td>Hemocytometer</td><td>Millicell</td><td>MDH-4N1</td><td></td></tr><tr><td>Hyaluronidase</td><td>Sigma-Aldrich</td><td>H4274</td><td></td></tr><tr><td>IBMX (3-Isobutyl-1-methylxanthine)</td><td>Selleck</td><td>S5836</td><td></td></tr><tr><td>KnockOut Serum Replacement</td><td>Gibco</td><td>A3181501</td><td></td></tr><tr><td>L-Cysteine</td><td>Sigma-Aldrich</td><td>C7352</td><td></td></tr><tr><td>Luteinizing hormone</td><td>Millipore</td><td>869003</td><td></td></tr><tr><td>M2 medium</td><td>Sigma-Aldrich</td><td>M7167</td><td></td></tr><tr><td>Medium 199</td><td>Gibco</td><td>11150-059</td><td></td></tr><tr><td>Micropipette</td><td>Gilson</td><td>F167350</td><td></td></tr><tr><td>Mineral oil&amp;nbsp;</td><td>Sigma-Aldrich</td><td>M8410</td><td></td></tr><tr><td>Phosphate-buffered saline</td><td>Gibco</td><td>20012027</td><td></td></tr><tr><td>Pregnant Mare Serum Gonadotropin</td><td>Ningbo Second hormone factory (Ningbo, China)</td><td>N/A</td><td></td></tr><tr><td>Sodium pyruvate</td><td>Sigma-Aldrich</td><td>P4562</td><td></td></tr><tr><td>Stereo microscope</td><td>Olympus</td><td>SZX10</td><td></td></tr><tr><td>Sterile medium bottles</td><td>Nalgene</td><td>46600-586</td><td></td></tr><tr><td>Sterile syringe needle</td><td>BD Biosciences</td><td>309623</td><td>1 mL, 27G&lt;sup&gt;1/2&lt;/sup&gt;</td></tr><tr><td>Syringe Filter Unit</td><td>Millipore</td><td>SLGP033R</td><td></td></tr><tr><td>Thermo Plate</td><td>Tokai Hit</td><td>TPi-SZX2X</td><td></td></tr></tbody></table>

mouse oocyte in vitro maturation, fertilization, and culture of preimplantation embryos

Mouse Oocyte In Vitro Maturation, Fertilization, and Culture of Preimplantation Embryos

Mouse oocyte in vitro maturation, combined with in vitro fertilization and subsequent embryo culture, enables the generation of preimplantation embryos under defined conditions. These procedures facilitate the investigation of the biological functions and molecular mechanisms underlying oocyte maturation, fertilization, and early embryo development. However, the successful application of this approach depends on careful handling of oocytes and sperm, appropriate culture conditions, and accurate assessment of developmental progression at each stage. Here, we describe methods for isolating immature cumulus&#8211;oocyte complexes (COCs) from mouse ovaries, in vitro maturation of oocytes, in vitro fertilization, and in vitro culture of embryos to the blastocyst stage. These procedures include preparing and equilibrating media and culture dishes, isolating and selecting COCs after ovarian dissection, collecting sperm and capacitating them, performing insemination under standardized conditions, and subsequently culturing embryos for developmental assessment. Developmental progression is evaluated using morphological criteria, including the formation of male and female pronuclei, cleavage to the two-cell stage, and blastocyst formation. This protocol can be integrated into studies of oocyte maturation and maternal factor function, providing a valuable tool for basic research in mammalian reproductive biology.

Using this protocol, mouse COCs were sequentially processed through in vitro maturation, in vitro fertilization, and subsequent in vitro embryo culture, resulting in reproducible generation of preimplantation embryos and development to the blastocyst stage under the conditions described. After 14 h of IVM, COCs typically showed cumulus expansion and intact oocyte morphology. Oocyte meiotic maturation was quantified by denuding a designated assessment cohort after IVM and scoring PB1 extrusion (...

This protocol describes procedures for in vitro maturation of mouse oocytes, in vitro fertilization, and subsequent embryo culture to the blastocyst stage.

Mouse oocyte in vitro maturation, combined with in vitro fertilization and subsequent embryo culture, enables the generation of preimplantation embryos ...

Our research focuses on controlled in vitro maturation and fertilization of mouse oocyte, an early embryonic development of pre-implantation embryos. Existing methods often separate in vitro maturation from in vitro fertilization in embryo culture. Our protocol integrated these steps and support efficient developmental rates.To begin, place the euthanized female C57BL/6 mouse aged 8 to 10 weeks in a supine position 48 hours after pregnant mare serum gonadotropin injection. Spray the abdomen with 70%ethanol to wet the fur and prevent hair from entering the abdominal cavity during ovary isolation. Use fine dissecting scissors to make a small lower abdominal skin incision and gently open the skin with gloved hands to expose the peritoneal membrane.After opening the peritoneal membrane and gently moving the intestines aside, locate the uterine horn and follow it toward the ovary. Identify the ovary near the kidney, typically enclosed by a fat pad. Use fine dissecting scissors to separate the ovary from the uterine horn and trim away excess connective tissue and fat.Immediately transfer the ovaries to pre-warmed M2 medium supplemented with Isobutyl methylxanthine or IBMX. Then collect the contralateral ovary and transfer it to the same dish. Place the dish under a stereo microscope and visualize the ovary prior to follicle puncture and cumulus-oocyte complexes or COCs collection.For COCs collection, gently puncture visible antral follicles with a sterile syringe needle to release COCs in the M2 medium. Use a finely pulled glass Pasteur pipette to collect the released COCs. Transfer them to a clean 35 millimeter dish containing two milliliters of pre-warmed M2 medium supplemented with 0.2 millimolar IBMX.Wash the COCs by serially moving them to clean areas of the dish, leaving detached granulosa cells and tissue debris behind. After debris removal, transfer the COCs to the in vitro maturation or IVM wash dish and wash them by sequential transfer through three 100 microliter drops of IVM wash medium. Transfer the washed COCs into 100 microliter drops of IVM medium with 20 to 30 COCs per drop.Culture them at 37 degrees Celsius in 5%carbon dioxide for 14 hours. Remove the cauda epididymis from a 10 to 16-week-old euthanized DBA2 male mouse avoiding fat, blood, and tissue fluid. Transfer both cauda epididymis into a 300 microliter drop of pre-equilibrated G-IVF medium for sperm release and capacitation.Immobilize the cauda epididymis using a pair of forceps. Make five to seven cuts in the cauda epididymis with a sterile syringe needle and shred the tissue to allow the release of concentrated sperm into the media. Incubate the dish at 37 degrees Celsius in 5%carbon dioxide for one hour to allow sperm capacitation.After 14 hours of in vitro maturation, wash the COCs by sequential transfer through three 100 microliter drops of in vitro fertilization or IVF wash medium. Transfer the washed COCs into the IVF insemination drops with 20 to 30 COCs per 100 microliter drop. Pre-incubate the fertilization dish containing COCs at 37 degrees Celsius in 5%carbon dioxide for 30 minutes before insemination.After capacitation, determine the sperm concentration using a hemocytometer. Based on the sperm concentration, calculate the required inoculum volume. Add capacitated sperm to each IVF drop to achieve a final concentration of four to 10 times 10 to the power of five sperm cells per milliliter.Incubate the IVF dishes containing COCs and sperm at 37 degrees Celsius in a 5%carbon dioxide incubator for four to six hours. After incubation, aspirate the presumptive zygotes with a finely pulled Pasteur pipette in minimal volume. Transfer them into the IVF wash drops.Gently pipette within each drop to remove excess sperm and debris. Wash the presumptive zygotes by sequential transfer through three IVF wash drops. Move the washed presumptive zygotes to the in vitro culture or IVC wash dish and sequentially transfer them through three pre-warmed embryo culture medium drops, minimizing IVF medium carryover.Place the presumptive zygotes into embryo culture medium with 20 to 30 embryos per 100 microliter drop. Culture them at 37 degrees Celsius in 5%carbon dioxide. After six to eight hours, assess fertilization by scoring the presence of male and female pronuclei, and discard oocytes that retain only one polar body and show no visible pronuclei.After 24 hours, check and count the number of two-cell embryos and calculate the two-cell rate as the number of two-cell embryos divided by the number of inseminated oocytes. Next, remove and discard uncleaved embryos. Continue to culture the embryos under the same incubation conditions until 96 hours post-insemination.Assess blastocyst formation at 96 hours post-insemination based on morphological criteria. After 14 hours of in vitro maturation, COCs typically exhibit cumulus expansion and intact morphology, allowing quantification of myotic maturation by denuding a designated cohort and scoring first polar body or PB1 extrusion. The average maturation rate based on PB1 extrusion rates across three independent replicates was 81.3%Following insemination, fertilization was assessed after six to eight hours by scoring the formation of male and female pronuclei.Cleavage was evaluated 24 hours after insemination using the two-cell rate. The average two-cell rates across three independent replicates were 87.6%The developmental competence was evaluated by further culturing embryos in vitro, and by assessing blastocyst formation at 96 hours post-insemination. The average blastocyst formation rate across three independent replicates was 52.6%Poor quality cleavage outcomes include embryos that fail to cleave, fragmented embryos, or embryos with unevenly sized blastomeres.Suboptimal developmental outcomes during extended culture included arrest at the two-cell stage, poorly expanded blastocysts, or embryos with obvious fragmentation and abnormal morphology. Stable temperature, optimal kits, and careful handing are critical for successful embryonic development under controlling visual conditions. Embryos generated using this protocol can be utilized for imaging, gene expression analysis, vitrification, or embryo transfer.Researchers can use this protocol to evaluate the genetic, pharmacological, or counter-condition effects on embryonic development.

mouse-oocyte-vitro-maturation-fertilization-culture-preimplantation

Research

JoVE Journal

Developmental Biology

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Mouse Oocyte Microinjection, Maturation and Ploidy Assessment

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Culture and Co-Culture of Mouse Ovaries and Ovarian Follicles

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Functional Manipulation of Maternal Gene Products Using In Vitro Oocyte Maturation in Zebrafish

This manuscript describes an experimental approach to morphologically and biochemically characterize horse oocytes. Specifically, the present work illustrates how to collect immature and mature horse oocytes by ultrasound-guided ovum pick-up (OPU) and how to investigate chromosome segregation, spindle morphology, global histone acetylation, and mRNA expression.

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A Neural Network-Based Identification of Developmentally Competent or Incompetent Mouse Fully-Grown Oocytes

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Mouse Oocyte In Vitro Maturation, Fertilization, and Culture of Preimplantation Embryos

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