The authors are very grateful to: dr Waclaw Tworzydlo (Department of Developmental Biology and Invertebrate Morphology, Institute of Zoology and Biomedical Research, Jagiellonian University) for technical facilities in TEM; to Ms. Beata Snakowska (Department of Endocrinology, Institute of Zoology and Biomedical Research, Jagiellonian University) for technical assistance; to the Department of Cell Biology and Imaging, Institute of Zoology and Biomedical Research, Jagiellonian University, JEOL JEM 2100HT (JEOL, Tokyo, Japan). This work was supported by grant 2018/29/N/NZ9/00983 from National Science Centre Poland.

The following procedures were approved by the Animal Welfare Committee at the Institute of Zoology and Biomedical Research at Jagiellonian University.
1. Isolation of porcine cumulus-oocyte complexes
<ol>
	<li>To collect material for the isolation of ovarian follicles, excise porcine ovaries from prepubertal gilts (approximately 6-7 months of age, weighing 70 to 80 kg) at a local slaughterhouse. Choose approximately 20 pig ovaries from 10 animals for COCs isolation in each experiment. 
...

The ability to maintain in vitro growth not only of the oocyte but also of cumulus cells surrounding it and simultaneously supporting its maturation is exceedingly essential for the successful assisted reproductive technologies and for furthering the understanding of somatic cell/oocyte interactions especially in species undergoing prolonged follicular growth such as human beings or pigs. Continuously improved IVM techniques are becoming useful tool to preserve reproductive options in cases of polycystic ovarian syndrome...

The development of various culture systems, including three-dimensional (3D) ones, aims to provide optimal conditions for the growth and maturation of oocytes isolated from the follicles even at earliest stages of development. This is of great importance for assisted reproductive techniques (ART), especially in view of the increasing number of women who are struggling with infertility after cancer treatment1. Maturation of oocytes in in vitro conditions (IVM) is already a well-established technique mainly used for in vitro embryo generation for the purpose of livestock reproduction2. However, in most mammalian species, even if high rates of maturation of cumulus-oocyte complexes (COCs) can be achieved (range 60 to 90&#160;%)3, their developmental competence is still inadequate to the needs. This is because the development of the zygotes obtained in such a way even up to the blastocyst stage is low and after the transfer into surrogate animals their viability to term is reduced. Consequently, there is a need to increase the developmental competence of embryos obtained from oocytes that were subjected to the IVM procedure4. Therefore, new maturation media5 are being devised and various periods of in vitro culture are tested6,7 along with supplementation of culture media with various growth factors and molecules8,9.
The first step of any complete IVM system is to create optimal conditions for sustainable growth of oocytes during in vitro culture. The oocyte growth is one of the specific indicators of the oocyte&#39;s ability to resume meiosis10,11. In addition, an appropriate oocyte in vitro culture system must be capable of supporting its nuclear maturation and cytoplasmic differentiation12. The morphology of the cumulus-oocyte complex is another important indicator used in ART clinics to select the best oocyte for subsequent steps of in vitro fertilization (IVF) procedure in humans and livestock12,13. Among morphological characteristics of COCs considered are: the oocyte diameter, its cytoplasm granulation and the first polar body integrity14,15. Besides, the oocyte developmental potential is correlated to the appearance and compaction of cumulus cells and number of their layers surrounding the oocyte. Very important for the appropriate oocyte in vitro culture system is also the maintenance of oocyte&#8211;cumulus cells proper interactions and cytoskeleton stability16,17,18,19. So far, in vitro oocyte growth within human COCs has been demonstrated20. The use of cow COCs also resulted with live births. These were isolated from immature ovarian follicles and then cultured for 14 days until the oocyte was sufficiently large to undergo the IVF procedure21. Similarly, COCs isolated from baboon antral follicles, subjected to IVM after in vitro culture yielded oocytes capable of reinitiating meiosis to the metaphase II stage with a normal appearing spindle structure22. However, in this study the authors did not try to fertilize them. Nevertheless such results indicate that a similar procedure could be applied not only to these particular mammalian species but also to human cumulus-oocyte complexes obtained from follicles what should allow to obtain oocytes of good quality suitable for a successful IVF technique.
The above described results were obtained with the application of conventional IVM protocols during which oocytes were cultured in two-dimensional (2D) systems. The routine procedure in 2D culture systems is covering oocytes, immersed in a drop of an appropriate culture media, with mineral oil23,24. It is assumed that an oil overlay during in vitro oocyte culture serves to prevent liquid evaporation, thus ensuring the maintenance of proper pH and osmotic pressure in the culture. Although such a 2D culture system allows to obtain, even up to 87% of mature pig oocytes25, it has been proven that the mineral oil overlay causes substantial diffusion of lipid soluble materials which are necessary for proper oocytes development26. Additionally, because of steroids (progesterone and estrogens) diffusion into the mineral oil during oocyte culture, a delay of nuclear maturation and reduction in developmental competence achievement of pig oocytes was observed. This may result in obtaining a small number of zygotes, which additionally are characterized by low developmental capacity to the stage of the blastocyst and by poor viability after transfer into recipient animals27. Therefore, attempts are being made to increase the developmental competence of embryos derived from oocytes received after IVM procedure, by creating optimal conditions for achieving both cytoplasmic and nuclear maturity of oocytes cultured together with CCs as complexes, especially using three-dimensional (3D) systems. Various innovative 3D in vitro culture systems have been developed in the last two decades28,29. These were designed to maintain the natural spatial organization of cells and to avoid their flattening in culture dishes what cannot be achieved in the traditional 2D cultures. The structural and functional activity of cultured COCs can be ensured by the maintenance of their proper architecture and undisturbed communication through gap-junctions between various compartments30. The suitability of bio-scaffolds for 3D in vitro culture of cumulus&#8211;oocyte complexes has been evaluated using natural biomaterials such as various components of the extra-cellular matrix (ECM; collagen and hyaluronic acid)31 or inert polymers (alginate)32. These attempts tested in several species brought promising results in terms of oocyte meiosis resumption and achievement of their full competence33,34,35. However so far, no 3D system suitable for COCs maturation isolated from large domestic animals, including pigs, has been developed.
This work describes two protocols that can be used for the 3D culture of porcine COCs. The first protocol describes encapsulation in fibrin-alginate beads (FAB). FAB can be formed by simultaneous mixing an alginate and fibrin solution, which undergo a synchronous gelation process. This combination provides a dynamic mechanical environment because both components contribute to matrix rigidity. A similar solution has been used previously for mouse ovarian follicle culture and maturation36. In the case of the presented protocol, to avoid premature degradation of the alginate-fibrin network, appropriately higher concentrations of calcium chloride solution are used, ensuring a fast and stable gelation process. The dynamic mechanical environment creates conditions similar to these in the natural intra-follicular environment in which COCs reside and increase in size. Additionally, the work shows representative results of COCs 3D culture systems, in which these are suspended in a drop of medium and encapsulated with fluorinated ethylene propylene (FEP; a copolymer of hexafluoropropylene and tetrafluoroethylene) powder particles, to form microbioreactors (Liquid Marbles, LM). LM are a form of 3D bioreactor that have been previously shown to support, among others, growth of living microorganisms37, tumor spheroids38 and embryonic stem cells39. LMs have been also successfully used for sheep oocyte culture40. In most experiments using LMs, bioreactors were prepared using polytetrafluoroethylene (PTFE) powder bed with particle size of 1 &#956;m41. The presented protocol uses FEP, which is very similar in composition and properties to the fluoropolymers PTFE. But FEP is more easily formable and softer than PTFE and what is especially important, it is highly transparent.
Both 3D systems maintain the gaseous in vitro culture environment. They also maintain COCs 3D organization by preventing their flattening and consequent disruption of gap junctions, preserving their functional relationship between the oocyte and surrounding follicular cells.

<table border="0" cellpadding="0" cellspacing="0" style="width:761px;" width="761">
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		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">General</td>
			<td style="width:139px;"></td>
			<td style="width:162px;"></td>
			<td style="width:204px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:256px;">Antibiotic Antimycotic (100x) 100ml</td>
			<td style="width:139px;">Thermo Fisher</td>
			<td>15240062</td>
			<td style="width:204px;">2.5% final concentration for Handling Medium. 1% in PBS (step 1.2)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:256px;">DMEM/F12 (500ml)</td>
			<td style="width:139px;">Sigma-Aldrich</td>
			<td>D8062</td>
			<td style="width:204px;">Handling and Maturation Medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">DPBS (w/o Ca, Mg), 1x, 500ml</td>
			<td style="width:139px;">Thermo Fisher</td>
			<td>14190144</td>
			<td style="width:204px;"></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:256px;">FCS (100 ml)</td>
			<td style="width:139px;">Thermo Fisher</td>
			<td>16140063</td>
			<td style="width:204px;">10% final concentration for both Handling Medium and Maturation Medium. (steps: 1.5. 2.6.)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">PBS (1x, pH 7.4) 500ml</td>
			<td style="width:139px;">Thermo Fisher</td>
			<td>10010023</td>
			<td style="width:204px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">TBS Stock Solution (10x, pH 7.4) 500 ml</td>
			<td>Cayman Chemicals</td>
			<td>600232</td>
			<td style="width:204px;">1x final concentration. Other brand can be use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">Maturation Medium</td>
			<td style="width:139px;"></td>
			<td style="width:162px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">hCG (1 VIAL of 10 000 U)</td>
			<td style="width:139px;">Sigma-Aldrich</td>
			<td style="width:162px;">CG10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">PMSG</td>
			<td style="width:139px;">BioVendor</td>
			<td>RP1782721000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">Fibrin-alginate beads</td>
			<td style="width:139px;"></td>
			<td style="width:162px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">Alginate Lyase</td>
			<td style="width:139px;">Sigma-Aldrich</td>
			<td style="width:162px;">A1603</td>
			<td>(Step 2.11.1)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">Thrombin</td>
			<td style="width:139px;">Sigma-Aldrich</td>
			<td>T9326-150UN</td>
			<td>(Step 2.1)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">Calcium Chloride</td>
			<td style="width:139px;">Sigma-Aldrich</td>
			<td style="width:162px;">C5670</td>
			<td>(Step 2.1)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">Fibrinogen (250mg)</td>
			<td style="width:139px;">Sigma-Aldrich</td>
			<td style="width:162px;">F3879</td>
			<td>(Step 2.2)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:256px;">Sodium Alginate</td>
			<td style="width:139px;">Sigma-Aldrich</td>
			<td>W201502</td>
			<td style="width:204px;">(Step 2.3) use for alginate solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">Liquid Marble</td>
			<td style="width:139px;"></td>
			<td style="width:162px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:256px;">FEP</td>
			<td style="width:139px;">Dyneon GmbH 3M AdMD</td>
			<td style="width:162px;">A-66670</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">Morphological examination</td>
			<td style="width:139px;"></td>
			<td style="width:162px;"></td>
			<td></td>
		</tr>
		<tr height="105">
			<td height="105" style="height:104px;width:256px;">LIVE/DEAD Viability/Cytotoxicity Kit, for mammalian cells</td>
			<td style="width:139px;">Thermo Fisher</td>
			<td>L3224</td>
			<td style="width:204px;">(Step 4.1.) Emitted fluorescence: 494 nm for calcein, 528 nm for EthD-1; measure: 517 nm for calcein, 617 nm - EthD-1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">VECTASHIELD Antifade Mounting Medium</td>
			<td style="width:139px;">Vector Laboratories</td>
			<td>H-1000</td>
			<td>mounting medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">Ultrastructure examination</td>
			<td style="width:139px;"></td>
			<td style="width:162px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:256px;">Glutaraldehyde solution</td>
			<td style="width:139px;">Sigma-Aldrich</td>
			<td style="width:162px;">G5882</td>
			<td style="width:204px;">2.5% final concentraion (Step 4.2.1.)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">LR White resin</td>
			<td style="width:139px;">Sigma-Aldrich</td>
			<td style="width:162px;">L9774</td>
			<td>(Step 4.2.4.)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">Methylene blue</td>
			<td style="width:139px;">Sigma-Aldrich</td>
			<td style="width:162px;">M9140</td>
			<td>(Step 4.2.5.)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">Osmium Tetroxide</td>
			<td style="width:139px;">Sigma-Aldrich</td>
			<td style="width:162px;">O5500</td>
			<td>(Step 4.2.3.)</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">Sodium cacodylate trihydrate</td>
			<td style="width:139px;">Sigma-Aldrich</td>
			<td style="width:162px;">C0250</td>
			<td style="width:204px;">Use for preparing 0.1M sodium cacodylate buffer (pH 7.2)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">Uranyl Acetate</td>
			<td style="width:139px;">POCH</td>
			<td>868540111</td>
			<td>(Step 4.2.4.)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Specific instruments, tools</td>
			<td style="width:139px;"></td>
			<td style="width:162px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">30 mm Pteri dish</td>
			<td style="width:139px;">TPP</td>
			<td>93040</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">60 mm IVF Petri dish</td>
			<td style="width:139px;">Falcon</td>
			<td>353653</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:256px;">Ez-Grid Premium Cell Handling Pipettor</td>
			<td style="width:139px;">RI Life Sciences</td>
			<td style="width:162px;">8-72-288</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">Ez-Tip</td>
			<td style="width:139px;">RI Life Sciences</td>
			<td style="width:162px;">8-72-4155/20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">Heating Table</td>
			<td style="width:139px;">SEMIC</td>
			<td style="width:162px;"></td>
			<td>Other brands can be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">Incubator</td>
			<td style="width:139px;">Panasonic</td>
			<td style="width:162px;">MCO-170AIC-PE</td>
			<td>Other brands can be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">Sterile petri dish (10 cm)</td>
			<td>NEST Biotechnology</td>
			<td>704002</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:256px;">Sterile syringe filters with 0.2 &#181;m</td>
			<td style="width:139px;">GOOGLAB SCIENTIFIC</td>
			<td>GB-30-022PES</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">Thermos</td>
			<td style="width:139px;">Quechua</td>
			<td>5602589</td>
			<td>Other brands can be used</td>
		</tr>
	</tbody>
</table>

proteolytically degraded alginate hydrogels and hydrophobic microbioreactors for porcine oocyte encapsulation

In reproductive biology, the biotechnology revolution that began with artificial insemination and embryo transfer technology led to the development of assisted reproduction techniques such as oocyte in vitro maturation (IVM), in vitro fertilization (IVF) and cloning of domestic animals by nuclear transfer from somatic cell. IVM is the method particularly of significance. It is the platform technology for the supply of mature, good quality oocytes for applications such as reduction of the generation interval in commercially important or endangered species, research concerning in vitro human reproduction, and production of transgenic animals for cell therapies. The term oocyte quality includes its competence to complete maturation, be fertilized, thereby resulting in healthy offspring. This means that oocytes of good quality are paramount for successful fertilization including IVF procedures. This poses many difficulties to develop a reliable culture method that would support growth not only of human oocytes but also of other large mammalian species. The first step in IVM is the in vitro culture of oocytes. This work describes two protocols for the 3D culture of porcine oocytes. In the first, 3D model cumulus-oocyte complexes (COCs) are encapsulated in a fibrin-alginate bead interpenetrating network, in which a mixture of fibrin and alginate are gelled simultaneously. In the second one, COCs are suspended in a drop of medium and encapsulated with fluorinated ethylene propylene (FEP; a copolymer of hexafluoropropylene and tetrafluoroethylene) powder particles to form microbioreactors defined as Liquid Marbles (LM). Both 3D systems maintain the gaseous in vitro culture environment. They also maintain COCs 3D organization by preventing their flattening and consequent disruption of gap junctions, thereby preserving the functional relationship between the oocyte, and surrounding follicular cells.

In COCs using both IVM systems, the granulosa cells adhered tightly to each other, and most of the recovered COCs had intact layers of cumulus cells (Figure 1A,B). Additionally, substantial proportion of cumulus cells were retained.
The results obtained from the COCs viability analysis confirmed that both systems applied for the encapsulation of porcine oocytes in 3D in vitro conditions ensured optimal growth conditions (Figur...

Watch this Scientific Journal Video about Proteolytically Degraded Alginate Hydrogels and Hydrophobic Microbioreactors for Porcine Oocyte Encapsulation at JoVE.com

Proteolytically Degraded Alginate Hydrogels and Hydrophobic Microbioreactors for Porcine Oocyte Encapsulation

Presented here are two protocols for the encapsulation of porcine oocytes in 3D culture conditions. In the first, cumulus-oocyte complexes (COCs) are encapsulated in fibrin-alginate beads. In the second, they are enclosed with fluorinated ethylene propylene powder particles (microbioreactors). Both systems ensure optimal conditions to maintain their 3D organization.

In reproductive biology, the biotechnology revolution that began with artificial insemination and embryo transfer technology led to the development of ...

This protocol for encapsulation of porcine oocytes makes it possible to maintain spherical organization of COCs. This prevents their flattening and consequent disruption of gap junctions between the oocyte and surrounding follicular cells. The main advantages of the two described protocols are that are user-friendly and allow for effective control of both the oocyte and chemo cell survival.Both of the culture systems are valuable tools for basic research in reproductive biology, and may have clinical relevance for improved infertility treatment. They can also be applied for developing novel biotechnology methods and for livestock improvement. After rinsing the ovaries, transfer them to a beaker filled with HM medium and store them in an incubator at 38 degrees Celsius during all subsequent manipulations.Aspirate the follicular fluid from large porcine follicles and centrifuge it at 100 times G for 10 minutes at room temperature. Then, filter the supernatant using a sterile syringe attached to a 0.2-micrometer membrane pore filter, and snap freeze it at negative 80 degrees Celsius. To isolate the COCs from medium-sized follicles, transfer two to three ovaries to a sterile 10 centimeter Petri dish filled with HM.Gently cut the surface of the protruding ovarian follicles with a sterile surgical number 15 blade, which will cause the follicular fluid and the COCs to flow out into the Petri dish.Aspirate the follicular contents with a 28 gauge needle attached to a disposable syringe, and transfer it into another Petri dish. To prepare the IVF Petri dishes, add one milliliter of HM to the central wells, and place three to four drops of HM in the outer rings. Then use a polycarbonate micropipette to move the undamaged COCs to drops of HM in the outer rings, and briefly rinse them three to four times.When finished, individually transfer them into the central well. Store the IVF plates in the incubator. Prepare thrombin and fibrinogen solutions as described in the text manuscript.On the day of the procedure, slowly thaw the fibrinogen solution on ice, and bring it to room temperature right before use. Mix 0.5%alginate solution and the fibrinogen solution at a one to one ratio for a final volume of two milliliters and gently vortex the mixture. Repair incubation chambers by applying thin strips of paraffin film to glass microscope slides.Pipette 7.5 microliter drops of the FA mixture onto the paraffin film-coated glass slide with separating spacers, placing eight to 10 drops arranged in two rows. Use a micropipette to transfer three to five COCs to the center of the FA drop, along with a minimum volume of maturation medium. Add 7.5 microliters of thrombin solution on top of each FA drop to cover it.There's no need to mix them because the gel forms almost instantaneously. Cover the incubation chamber with a previously prepared glass slide, then turn the chamber upside down and place it in a 100-millimeter Petri dish lined with moist filter paper. Put the Petri dish in the 5%carbon dioxide at 38 degrees Celsius incubator for five to seven minutes.After incubation, transfer each FA capsule into a well of a 96-well plate containing 100 microliters of maturation medium. Every two days, replace half of the medium with fresh pre-equilibrated maturation medium. Image COC is using an inverted light microscope at 10x magnification.After culturing the COCs for four days, remove the maturation medium from the wells and add 100 microliters of 10 international units per milliliter algenate lysate in DMEM. Leave the culture plate in the incubator for 25 to 30 minutes. Remove the COCs from the dissolved capsules.Wash them in DMEM several times, then transfer them to the inner ring of an IVF dish containing PBS. Make a bed of five grams of FEP powder in a 30 millimeter Petri dish. Distribute a single droplet of maturation medium containing three to five COCs onto the FEP powder.Rotate the plate gently in a circular motion to ensure that the particles completely cover the surface of the liquid drop and form liquid marbles, or LMs. Prepare several 60 millimeter IVF Petri dishes, and add three to four milliliters of sterile water to the outer rings to create a humidity chamber. Pick up the formed LM with a 1000 microliter pipette and place it into the central well.Incubate the marbles for four days at 38 degrees Celsius in a five percent carbon dioxide incubator. To change the medium, apply 30 microliters of maturation medium onto each LM, which will cause it to spread. When the marble contents dissolve, transfer the COCs released from the bio-reactor to a drop of fresh maturation medium in the Petri dish.After three to four washes in maturation medium, transfer the COCs, along with 30 microliters of fresh medium, onto the FEP powder bed. Gently rotate the plate in a circular motion to ensure that the powder particles completely cover the surface of the liquid drop and form a new LM.Then, transfer the LM to the IVF Petri dish. Both in vitro maturation systems produced COCs with granulosis cells that were tightly adhered to each other and intact layers of cumulus cells.The COC viability analysis confirmed that optimal growth conditions were achieved in both encapsulation systems. In both groups, only high viability oocytes sites were observed. The oocytes were imaged with transmission electron microscopy.The mitochondria were evenly distributed and had a shell-like shape. Only a few of them were elongated and their clustering was sporadically observed. Endoplasmic reticulum was either associated with mitochondria or free in the oocyte cytoplasm.Liquid droplets appeared as small, dark, round structures, and Golgi apparatus were emerged with dilated cisternae. It is important to be precise and careful when transferring FA capsules from incubation chamber into culture plates and when picking up and transferring format LM into the IVF Petri dish.

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5.3K Views.  Jagiellonian University in Krakow. This protocol for encapsulation of porcine oocytes makes it possible to maintain spherical organization of COCs. This prevents their flattening and consequent disruption of gap junctions between the oocyte and surrounding follicular cells. The main advantages of the two described protocols are that are user-friendly and allow for effective control of both the oocyte and chemo cell survival.Both of the culture systems are valuable tools for basic research in reproductive biology, and ma...