This work was partly supported by EVSSAR (European Veterinary Society for Small Animal Reproduction) Grant 2016 and by Universit&#224; degli Studi di Milano, Piano di Sostegno alla Ricerca 2019 (Linea 2 Azione A). We also wish to thank Dr. MariaGiorgia Morselli for her contribution to the experiments hereby depicted and to image acquisition.

The procedures hereby depicted did not undergo ethical approval since cat ovaries were collected at veterinary clinics as byproducts from owner-requested routine ovariectomy or ovariohysterectomy.
1. Oocyte collection
<ol>
	<li>Before starting the experiments, prepare solutions for ovary and oocyte collection. Prepare ovary collection solution with phosphate buffered saline (PBS) with a mixture of antibiotics and antimycotics (100 IU/mL of penicillin G sodium, 0.1 mg/mL of streptomycin sulphate, 0.25 &#956;g/mL of amphotericin B). Prepare oocyte collection solution (i.e., PBS/PVA) with PBS with 100 IU/mL of penicillin G sodium, 0.1 mg/mL of streptomycin sulphate and 0.1% (w/v) polyvinyl alcohol (PVA).</li>
	<li>Store solutions for ovary and oocyte collection at 4 &#176;C until use.</li>
	<li>On the day of queens&#8217; spaying, a few hours before processing the samples, take part of ovary and oocyte collection solutions and let them warm up at room temperature (RT; 25 &#177; 2 &#176;C).</li>
	<li>When samples arrive to the lab, isolate the ovaries from the surrounding connective tissue and from the oviduct and wash them in fresh ovary collection medium in a Petri dish.</li>
	<li>Fill one 35 mm Petri dish for each queen with about 3 mL of RT PBS/PVA, and one more dish to collect the oocytes.</li>
	<li>Place a pair of ovaries in a Petri dish and mince the cortex with a surgical scalpel. Ensure all the follicles have been broken with the help of a stereomicroscope (magnification 8x).</li>
	<li>Collect COCs with a pipette and move them in fresh PBS/PVA in the allocated Petri dish. Select good qualities COCs, with a homogenous, dark cytoplasm and surrounded by several compact layers of cumulus cells (grade I18).</li>
</ol>
2. Vitrification
<ol>
	<li>Prepare equilibration and vitrification media.
	<ol>
		<li>Prepare equilibration solution (ES) with 7.5% (v/v) ethylene glycol (EG) and 7.5% (v/v) dimethylsulphoxide (DMSO) in Medium 199, with 20% fetal bovine serum (FBS).</li>
		<li>Prepare vitrification solution (VS) with 15% (v/v) EG, 15% (v/v) DMSO and 0.5 M sucrose in Medium 199, with 20% FBS (modified from 19).</li>
		<li>Let ES and VS warm up at RT before use. 
		NOTE: Cryoprotectants (e.g., EG, DMSO) are known to be cytotoxic. A strategy to counter their toxicity is to reduce the temperature at which the cells are exposed to them20, and oocytes are usually exposed to cryoprotectants at RT. Thus, the whole procedure, from oocyte collection to vitrification, is carried out at RT in this protocol to avoid temperature fluctuations.</li>
	</ol>
	</li>
	<li>Prepare the vitrification dish (i.e., a special dish consisting of six conical wells divided in two rows, known as &#8220;Repro plate&#8221;). Prepare one row (three wells) for each vitrification support.
	<ol>
		<li>Add 20 &#181;L of ES on the bottom of the first well.</li>
		<li>Add 300 &#181;L of VS on the bottom of the second (i.e., 1VS) and third (i.e., 2VS) wells.</li>
	</ol>
	</li>
	<li>Equilibrate COCs in ES.
	<ol>
		<li>With a small-bore pipette (e.g., a Pasteur pipette pulled on a Bunsen beak to make it thinner &#8211; diameter should be at least 200 &#181;m), transfer one (or more) COC(s) on the bottom of the first well. 
		NOTE: Choose the size of the pipette according to the number of layers of cumulus cells surrounding the oocytes, so to COCs dimensions, aiming to reduce as much as possible the volume of media which is transferred with COCs in each step. With the present protocol, trained operators can vitrify successfully up to eight feline COCs per each vitrification support.</li>
		<li>Slowly add 20 &#181;L of ES on the border of the drop. Wait for 3 minutes.</li>
		<li>Slowly add other 20 &#181;L of ES on the border of the drop. Wait for 3 minutes.</li>
		<li>Slowly add 240 &#181;L of ES on the border of the drop. Wait for 9 minutes.</li>
		<li>While waiting, prepare a box with liquid nitrogen (LN2), label a vitrification support with the experiment/cat identification code and leave it open. Put both of them near the stereomicroscope, so that they can be easily reachable while working. 
		CAUTION! Wear personal protective equipment when handling LN2.</li>
		<li>Alternatively, if several COCs have to be vitrified, begin the first equilibration step (see step 2.3.1 - 2.3.2) for another group of COCs (which will be loaded onto a new vitrification support).</li>
	</ol>
	</li>
	<li>Vitrify COCs in VS in less than 90 seconds.
	<ol>
		<li>Using the same small-bore pipette, fill it with 1VS, take the COCs from the bottom of the first well (ES) and move them to the surface of the second well (1VS).</li>
		<li>Wash the pipette with 1VS.</li>
		<li>Take the COCs and move them in another area (on the bottom) of the well; mix the medium surrounding them with the pipette.</li>
		<li>Fill the pipette with 1VS in another area of the well, take the COCs, move them and mix the medium surrounding them with the pipette.</li>
		<li>Repeat step 2.4.4.</li>
		<li>Wash the pipette with 2VS.</li>
		<li>Take the COCs and move them on the bottom of the third well (2VS); mix the medium surrounding them with the pipette.</li>
		<li>Fill the pipette with 2VS in another area of the well, take the COCs, move them and mix the medium surrounding them with the pipette.</li>
		<li>Repeat step 2.4.8.</li>
		<li>Fill the pipette with 2VS in another area of the well, take the COCs and load them on the strip of the vitrification support, as close as possible to the tip. Aspirate excess medium to reduce the volume of VS as much as possible.</li>
		<li>Immediately plunge the vitrification support in LN2, moving it. Close it with the help of some clamps, making sure it always remains immersed in LN2. 
		NOTE: Keeping the right timing is crucial due to cryoprotectant cell-toxicity. Because of their high concentration in vitrification media, cell exposure needs to be controlled, also by flawless execution of the technique21. If samples are abundant, consider dividing them to process them more quickly and minimize the amount of time from oocyte collection to vitrification.</li>
	</ol>
	</li>
	<li>Store the loaded vitrification supports in a goblet and keep them in a storage LN2 tank until warming. 
	NOTE: All the previous steps of the protocol can also be applied in field conditions if LN2 is available. A dry shipper will be necessary to transport the samples to the lab safely and then proceed there with the following phases.</li>
</ol>
3. Warming
<ol>
	<li>Prepare warming media.
	<ol>
		<li>Prepare thawing solution (TS) with 1 M sucrose in Medium 199, with 20% FBS.</li>
		<li>Prepare dilution solution (DS) with 0.5 M sucrose in Medium 199, with 20% FBS.</li>
		<li>Prepare washing solution (WS) with Medium 199 with 20% FBS.</li>
		<li>Before use, warm up TS to 38 &#176;C and DS and WS at RT.</li>
	</ol>
	</li>
	<li>If necessary, prepare the culture medium for the subsequent use of warmed COCs (e.g., in vitro maturation, IVM).</li>
	<li>Place the heating stage close to the stereomicroscope and turn it on (38 &#176;C). Put the lid of a Petri dish to warm up.</li>
	<li>When everything is ready, transfer the vitrification supports which must be warmed from the storage tank to a box with LN2, and put the box near the stereomicroscope.</li>
	<li>Prepare the &#8220;Repro plate&#8221;. Prepare a row for each vitrification support that has to be warmed.
	<ol>
		<li>Add 300 &#181;L of DS on the bottom of the first well.</li>
		<li>Add 300 &#181;L of WS on the bottom of the second (i.e., 1WS) and third (i.e., 2WS) wells.</li>
	</ol>
	</li>
	<li>Make a drop of TS (100 &#181;L) on the lid of the Petri dish.</li>
	<li>With clamps, open one vitrification support in the LN2.</li>
	<li>Put the TS drop under the stereomicroscope and with one fast movement take the vitrification support from the LN2 and immerse its strip in the drop, moving it until all COCs detach. Remove the support from the drop as soon as it is empty, but leave the COCs in TS for 1 minute in total (from immersion until the following step).</li>
	<li>Take a pipette similar to that used for vitrification and fill it with DS. Take the COCs from the drop (TS) and move them to the bottom of the first well (DS). Wait for 3 minutes.</li>
	<li>Wash the pipette with 1WS. Take the COCs and move them on the bottom of the second well (1WS). Wait for 5 minutes.</li>
	<li>Wash the pipette with 2WS. Take the COCs and move them on the surface of the third well (2WS). Wait for them to touch the bottom of the well.</li>
	<li>Repeat step 3.11 using another area of the well.</li>
	<li>Wash the pipette with culture medium and move the oocytes to the culture dish for the following use (e.g., IVM).</li>
</ol>

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<li>Colombo, M., Morselli, M. G., Apparicio, M., Luvoni, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Granulosa+cells+in+three-dimensional+culture%3A+A+follicle-like+structure+for+domestic+cat+vitrified+oocytes%5BTitle%5D)+AND+%22Reproduction+in+Domestic+Animals%22%5BJournal%5D)">Granulosa cells in three-dimensional culture: A follicle-like structure for domestic cat vitrified oocytes.</a> Reproduction in Domestic Animals. , (2020).</li>
<li>Wood, T. C., Wildt, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effect+of+the+quality+of+the+cumulus-oocyte+complex+in+the+domestic+cat+on+the+ability+of+oocytes+to+mature%2C+fertilize+and+develop+into+blastocysts+in+vitro%5BTitle%5D)+AND+%22Journal+of+Reproduction+and+Fertility%22%5BJournal%5D)">Effect of the quality of the cumulus-oocyte complex in the domestic cat on the ability of oocytes to mature, fertilize and develop into blastocysts in vitro.</a> Journal of Reproduction and Fertility. 110 (2), 355-360 (1997).</li>
<li>Cobo, A., Kuwayama, M., Pérez, S., Ruiz, A., Pellicer, A., Remohí, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comparison+of+concomitant+outcome+achieved+with+fresh+and+cryopreserved+donor+oocytes+vitrified+by+the+Cryotop+method%5BTitle%5D)+AND+%22Fertility+and+Sterility%22%5BJournal%5D)">Comparison of concomitant outcome achieved with fresh and cryopreserved donor oocytes vitrified by the Cryotop method.</a> Fertility and Sterility. 89 (6), 1657-1664 (2008).</li>
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<li>Brambillasca, F., Guglielmo, M. C., Coticchio, G., Mignini Renzini, M., Dal Canto, M., Fadini, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+current+challenges+to+efficient+immature+oocyte+cryopreservation%5BTitle%5D)+AND+%22Journal+of+Assisted+Reproduction+and+Genetics%22%5BJournal%5D)">The current challenges to efficient immature oocyte cryopreservation.</a> Journal of Assisted Reproduction and Genetics. 30 (12), 1531-1539 (2013).</li>
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<li>Calvo, J. J., Robert, D., Viqueira, M., Lombide, P., Calvo, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Vitrified+and+in+vitro+matured+oocytes+viability+from+adult+housecat+%28Felis+catus%29+in+reproductive+season%5BTitle%5D)+AND+%22International+Journal+of+Morphology%22%5BJournal%5D)">Vitrified and in vitro matured oocytes viability from adult housecat (Felis catus) in reproductive season.</a> International Journal of Morphology. 33 (4), (2015).</li>
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</ol>

The authors have nothing to disclose.

Oocyte cryopreservation is a crucial germplasm conservation technique, especially in taxa where many species are endangered, such as Felidae family. In this manuscript, a simple and field-friendly protocol for the vitrification of immature cat oocytes was presented. Laboratory-made media, minimum volume vitrification supports and trained personnel are the key factors for the success of this method, which allows obtaining viable oocytes consistently and repeatedly, as shown by the representative results hereby reported.</...

Cryopreservation has become a key step of assisted reproduction techniques (ARTs). In humans, it allows preservation of fertility or postponement of parenthood for medical or personal reasons. In animals, it is necessary to overcome distance and time in planned matings, especially in farm animals and pets, or to preserve genetic material of valuable subjects in conservation programs, particularly in wild endangered species. Gamete cryopreservation is the best choice when the individuals to be bred have not been chosen yet or in order to avoid ethical issues associated with embryo freezing, especially in human medicine1. Spermatozoa are relatively easy to preserve and give satisfactory outcomes after thawing, but oocytes, due to their structural features, might be more complex to store. Indeed, the low surface/volume ratio, as well as the presence of the zona pellucida surrounding the ooplasma, limits the movement of cryoprotectants and water across the cell2. Moreover, in domestic animals including felids, they are characterized by a lipid-rich cytoplasm, which is thought to make them more sensitive to cryopreservation3.
Most felids are threatened, and the domestic cat is used as a model to develop protocols for germplasm preservation thanks to the availability of gonads from routine ovariectomy. In wild animals, gonads can be obtained after elective surgeries or (more frequently) post-mortem, and immature (germinal vesicle) gametes can be retrieved. Hormonal stimulation aimed to obtain mature (metaphase II) oocytes is not as common as in human ARTs because of the ethical issues and the species- and individual-specific response to treatments4.
Therefore, the development of cryopreservation strategies has focused on immature gametes, which can usually be retrieved after the unexpected or sudden death of rare individuals. From a biological point of view, there are some differences in the cryopreservation of immature or mature gametes, each having its advantages. Firstly, DNA is more protected in immature oocytes, whose germinal vesicle contains chromosomes surrounded by a nuclear membrane, while the meiotic spindle of metaphase II oocytes could be more vulnerable to cryoinjuries5. Secondly, cold-induced cytoskeleton damages might affect spindle rotation, polar body extrusion, pronuclear migration and cytokinesis, which could have different impacts according to oocyte developmental phase, influencing meiosis progression or post-fertilization events. Finally, and perhaps most importantly, whereas mature oocytes are ready to be fertilized, immature gametes rely on the support of the surrounding cumulus cells to go through nuclear and cytoplasmic maturation6, and this is the reason why whole cumulus-oocyte complexes (COCs) are cryopreserved. However, the loss of cumulus cells and/or the loss of functional connection between the gamete and the surrounding somatic cells are probably the most detrimental effect of cryopreservation of immature COCs.
Among cryopreservation techniques, vitrification is one that can be applied more easily in field conditions. Compared to slow (or controlled rate) freezing, vitrification is faster and does not require specific equipment, such as a programmable freezer. In order to satisfy the three fundamental principles of vitrification (i.e., high viscosity, connected to high cryoprotectant concentration, small volumes and ultra-rapid temperature decrease), several media and supports especially have been developed and used in cats for both immature and mature oocytes. Beginning with simple straws7, devices were then developed to reach the &#8220;Minimum volume&#8221; goal. Cryoloop8, open pulled straws (OPS)9, plastic gutters (modified straw)10 and cryotubes11 have been used, until a more efficient device (i.e., Cryotop) was employed11, improving survival and meiosis resumption. Cryotop (Supplemental Figure 1) is a commercially available support which has become the elective open system for vitrification. Developed for the vitrification of human oocytes and embryos, it consists of a small film strip attached to a hard plastic holder, protected by a plastic tube cap during storage12. Thanks to its usability and to the extreme reduction in vitrification volume (as little as 0.1 &#181;L), which also leads to extremely rapid cooling and warming rates, this vitrification support has been increasingly applied in several species, including the domestic cat, in which it has been used with a variety of media13,14,15,16,17.
The purpose of this manuscript is to describe a collection-vitrification-warming protocol, with minor modifications from the one originally developed for human oocytes, which employs laboratory-made media and commercial supports for minimum volume vitrification and can be easily applied in field conditions for the cryopreservation of immature feline COCs.

<table><tbody><tr><td>Amphotericin B</td><td>Sigma-Aldrich</td><td>A2942</td><td>/</td></tr><tr><td>Automatic pipettes &amp;amp; tips</td><td>/</td><td>/</td><td>Needed to pipette 20, 100, 240 and 300 &amp;micro;L</td></tr><tr><td>Surgical scalpels</td><td>/</td><td>/</td><td>Size 10 is usually ok</td></tr><tr><td>Bunsen beak</td><td>/</td><td>/</td><td>/</td></tr><tr><td>Clamps</td><td>/</td><td>/</td><td>Some small (Mosquito clamp) for ovary isolation, some bigger (Klemmer clamp) to work in liquid nitrogen</td></tr><tr><td>Cryotop</td><td>Kitazato (distributor: MBT - Medical Biological Technologies)</td><td>01.CR</td><td>Distributors and catalog number may change in different countries</td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>Sigma-Aldrich</td><td>D2650</td><td>/</td></tr><tr><td>Ethylene glycol (EG)</td><td>Sigma-Aldrich</td><td>E9129</td><td>/</td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Sigma-Aldrich</td><td>F9665</td><td>/</td></tr><tr><td>Glass Pasteur pipettes</td><td>/</td><td>/</td><td>Advised lenght 230 mm (100+130)</td></tr><tr><td>Gobelets</td><td>/</td><td>/</td><td>According to the canisters of the storage tank</td></tr><tr><td>Heating stage</td><td>/</td><td>/</td><td>All heating stages are ok, as long as they can keep 38&amp;ordm;C</td></tr><tr><td>Liquid nitrogen</td><td>/</td><td>/</td><td>/</td></tr><tr><td>Medium 199</td><td>Sigma-Aldrich</td><td>M4530</td><td>/</td></tr><tr><td>Phosphate-buffered saline (PBS)</td><td>Sigma-Aldrich</td><td>D8662</td><td>/</td></tr><tr><td>Penicillin G sodium</td><td>Sigma-Aldrich</td><td>P3032</td><td>/</td></tr><tr><td>Polyvinyl alcohol (PVA)</td><td>Sigma-Aldrich</td><td>P8136</td><td>/</td></tr><tr><td>Repro plate</td><td>Kitazato (distributor: MBT - Medical Biological Technologies)</td><td>01.K-2</td><td>Distributors and catalog number may change in different countries</td></tr><tr><td>Stereomicroscope</td><td>/</td><td>/</td><td>As long as the operator can select the oocytes, other stereomicroscopes are ok</td></tr><tr><td>Storage tank</td><td>/</td><td>/</td><td>Any regularly filled tank is ok</td></tr><tr><td>Streptomycin sulphate</td><td>Sigma-Aldrich</td><td>S9137</td><td>/</td></tr><tr><td>Styrofoam/nitrogen resistant box</td><td>/</td><td>/</td><td>All boxes which can contain liquid nitrogen are ok, as long as the operator is comfortable. Kitazato box is called &quot;Cooling Rack&quot;</td></tr><tr><td>Sucrose</td><td>Sigma-Aldrich</td><td>S1888</td><td>/</td></tr><tr><td>Timers</td><td>/</td><td>/</td><td>All timers which can be set on times until 9 minutes are ok</td></tr></tbody></table>

minimum volume vitrification of immature feline oocytes

In wild animals&#8217; conservation programs, gamete banking is crucial to safeguard genetic resources of valuable individuals and rare species and to promote biodiversity preservation. In felids, most species are threatened with extinction, and domestic breeds are used as a model to increase the efficiency of protocols for germplasm banking. Among oocyte cryopreservation techniques, vitrification is more and more popular in human and veterinary assisted reproduction. Cryotop vitrification, which was at first developed for human oocytes and embryos, has demonstrated to be well-suited for cat oocytes. This method offers several advantages, such as the feasibility in field conditions and the speed of the procedure, which can be helpful when several samples need to be processed. However, the efficiency is strongly dependent on the operator&#8217;s skills, and intra- and inter-laboratory standardization are needed, as well as personnel training. This protocol describes minimum volume vitrification of immature feline oocytes on a commercial support in a step by step field-friendly protocol, from oocyte collection to warming. Following the protocol, preservation of oocyte integrity and viability at warming (as high as 90%) can be expected, although there is still room for improvement in post-warming maturation and embryonic development outcomes.

Following cat oocyte vitrification and warming according to the present protocol (Figure 1 and Supplemental Figure 1), the vast majority of gametes survive. After vitrification, among other techniques, viability can be evaluated at the optical microscope as morphological integrity22 or with the use of vital stains. One of the latter is fluorescein diacetate/propidium iodide (FDA/PI), which allows the identification of viable (bright green fluorescence...

Watch this Scientific Journal Video about Minimum Volume Vitrification of Immature Feline Oocytes at JoVE.com

Minimum Volume Vitrification of Immature Feline Oocytes

This manuscript describes a protocol for the minimum volume vitrification of immature cat oocytes with laboratory-made media on commercial supports. It covers every step from oocyte isolation from ex vivo gonads to vitrification and warming.

In wild animals&#8217; conservation programs, gamete banking is crucial to safeguard genetic resources of valuable individuals and rare species and to ...

minimum-volume-vitrification-of-immature-feline-oocytes

Research

JoVE Journal

Developmental Biology

6.4K Views.  Università degli Studi di Milano. Dit protocol is nuttig voor het behoud op lange termijn van de katachtige eicellen door middel van een cryopreservatie techniek bekend als met vitrification, die cruciaal is voor de vruchtbaarheid en de biodiversiteit behoud bij diersoorten. De belangrijkste voordelen van vitrificatie zijn de snelheid, omdat het kan worden voltooid in minder dan 17 minuten en de haalbaarheid ervan in het veld omstandigheden als vloeibare stikstof beschikbaar is. De huiskat is een levensvatbaar model voor wild...

Video: Minimum Volume Vitrification of Immature Feline Oocytes

Analysis of Chromosome Segregation, Histone Acetylation, and Spindle Morphology in Horse Oocytes

The field of assisted reproduction has been developed to treat infertility in women, companion animals, and endangered species. In the horse, assisted reproduction also allows for the production of embryos from high performers without interrupting their sports career and contributes to an increase in the number of foals from mares of high genetic value. The present manuscript describes the procedures used for collecting immature and mature oocytes from horse ovaries using ovum pick-up (OPU). These oocytes were then used to investigate the incidence of aneuploidy by adapting a protocol previously developed in mice. Specifically, the chromosomes and the centromeres of metaphase II (MII) oocytes were fluorescently labeled and counted on sequential focal plans after confocal laser microscope scanning. This analysis revealed a higher incidence in the aneuploidy rate when immature oocytes were collected from the follicles and matured in vitro compared to in vivo. Immunostaining for tubulin and the acetylated form of histone four at specific lysine residues also revealed differences in the morphology of the meiotic spindle and in the global pattern of histone acetylation. Finally, the expression of mRNAs coding for histone deacetylases (HDACs) and acetyl-transferases (HATs) was investigated by reverse transcription and quantitative-PCR (q-PCR). No differences in the relative expression of transcripts were observed between in vitro and in vivo matured oocytes. In agreement with a general silencing of the transcriptional activity during oocyte maturation, the analysis of the total transcript amount can only reveal mRNA stability or degradation. Therefore, these findings indicate that other translational and post-translational regulations might be affected.
Overall, the present study describes an experimental approach to morphologically and biochemically characterize the horse oocyte, a cell type that is extremely challenging to study due to low sample availability. However, it can expand our knowledge on the reproductive biology and infertility in monovulatory species.

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.

The field of assisted reproduction has been developed to treat infertility in women, companion animals, and endangered species. In the horse, assisted ...

Vitrification of In Vitro Matured Oocytes Collected from Adult and Prepubertal Ovaries in Sheep

In livestock, in vitro embryo production systems can be developed and sustained thanks to the large number of ovaries and oocytes that can be easily obtained from a slaughterhouse. Adult ovaries always bear several antral follicles, while in pre-pubertal donors the maximal numbers of oocytes are available at 4 weeks of age, when ovaries bear peak numbers of antral follicles. Thus, 4 weeks old lambs are considered good donors, even if the developmental competence of prepubertal oocytes is lower compared to their adult counterpart. 
Basic research and commercial applications would be boosted by the possibility of successfully cryopreserving vitrified oocytes obtained from both adult and prepubertal donors. The vitrification of oocyte collected from prepubertal donors would also allow shortening the generation interval and thus increasing the genetic gain in breeding programs. However, the loss of developmental potential after cryopreservation makes mammalian oocytes probably one of the most difficult cell types to cryopreserve. Among the available cryopreservation techniques, vitrification is widely applied to animal and human oocytes. Despite recent advancements in the technique, exposures to high concentrations of cryoprotective agents as well as chilling injury and osmotic stress still induce several structural and molecular alterations and reduce the developmental potential of mammalian oocytes. Here, we describe a protocol for the vitrification of sheep oocytes collected from juvenile and adult donors and matured in vitro prior to cryopreservation. The protocol includes all the procedures from oocyte in vitro maturation to vitrification, warming and post-warming incubation period. Oocytes vitrified at the MII stage can indeed be fertilized following warming, but they need extra time prior to fertilization to restore damage due to cryopreservation procedures and to increase their developmental potential. Thus, post-warming culture conditions and timing are crucial steps for the restoration of oocyte developmental potential, especially when oocyte are collected from juvenile donors.

The protocol aims at providing a standard method for the vitrification of adult and juvenile sheep oocytes. It includes all the steps from the preparation of the in vitro maturation media to the post-warming culture. Oocytes are vitrified at the MII stage using Cryotop to ensure the minimum essential volume.

In livestock, in vitro embryo production systems can be developed and sustained thanks to the large number of ovaries and oocytes that can be easily ...

Ovarian Tissue Oocyte-In Vitro Maturation for Fertility Preservation

Mature oocyte vitrification is the standard of care to preserve fertility in women at risk of infertility. However, ovarian tissue cryopreservation (OTC) is still the only option to preserve fertility in women who need to start gonadotoxic treatment urgently or in prepubertal children. During ovarian cortex preparation for cryopreservation, medullar tissue is removed. Growing antral follicles reside at the border of the cortex-medullar interface of the ovary and are broken during this process, releasing their cumulus-oocyte complex (COC). By thoroughly inspecting the medium and fragmented medullar tissue, these immature cumulus-oocyte complexes can be identified without interfering with the OTC procedure. The ovarian tissue-derived immature oocytes can be successfully matured in vitro, creating an additional source of gametes for fertility preservation. If OTC is performed within or near a medical assisted reproduction laboratory, all necessary in vitro maturation (IVM) and oocyte vitrification tools can be at hand. Furthermore, upon remission and child wish, the patient has multiple options for fertility restoration: ovarian tissue transplantation or embryo transfer after the insemination of vitrified/warmed oocytes. Hence, ovarian tissue oocyte-in vitro maturation (OTO-IVM) can be a valuable adjunct fertility preservation technique.

Ovarian Tissue Oocyte-In Vitro Maturation for Fertility Preservation

Presented here is ovarian tissue oocyte-in vitro maturation (OTO-IVM), an accessible technique within a medical assisted reproduction (MAR) laboratory offering realistic additional fertility preservation options to patients who need ovarian tissue cryopreservation.

Mature oocyte vitrification is the standard of care to preserve fertility in women at risk of infertility. However, ovarian tissue cryopreservation (OTC) ...

OP-IVM: Combining In vitro Maturation after Oocyte Retrieval with Gynecological Surgery

The use of in vitro maturation (IVM) before gynecological operation (OP-IVM) is an extension of conventional IVM that combines IVM following oocyte retrieval with routine gynecological surgery. OP-IVM is suitable for patients undergoing benign gynecological surgery who have the need for fertility preservation (FP) or infertility treatments such as in vitro fertilization and embryo transfer (IVF-ET). In the operating room, patients undergoing benign gynecological surgery are first anesthetized and receive ultrasound-guided immature follicle aspiration (IMFA) treatment. As the subsequent gynecological surgery is performed, the cumulus-oocyte complexes (COCs) are examined, and the immature COCs are transferred into the IVM medium and cultured for 28-32 hours in the IVF laboratory. After assessment, mature oocytes in the MII stage will be selected and cryopreserved in liquid nitrogen for FP or fertilized by intracytoplasmic sperm injection (ICSI) for IVF-ET. By combining IVM with gynecological surgery, immature oocytes that would have been discarded can be saved and used for assisted reproductive technology (ART). The procedure, significance and critical aspects of OP-IVM are described in this article.

In vitro maturation (IVM) before gynecological operation (OP-IVM) combines IVM following oocyte retrieval with routine gynecological surgery and serves as an extension of conventional IVM applications for fertility preservation.

The use of in vitro maturation (IVM) before gynecological operation (OP-IVM) is an extension of conventional IVM that combines IVM following oocyte ...

Medicine

Cryopreservation of Oocytes Retrieved from Ovarian Tissue to Optimize Fertility Preservation in Prepubertal Girls and Women

Human ovarian tissue cryopreservation (OTC) is increasingly used worldwide to preserve female fertility in prepubertal girls and women at risk of premature ovarian insufficiency (POI) in the context of urgent gonadotoxic treatments or ovarian surgery. Fertility preservation is challenging because there is no consensus regarding patient management, preservation fertility strategies, or even technical laboratory protocols, which implies that each procedure must be adapted to the characteristics of the patient profile and its own risk-benefit ratio. During OTC, mature/immature oocytes can be aspirated directly from large/small antral follicles within ovarian tissue samples and/or be released into culture media from growing follicles during ovarian tissue dissection in prepubertal girls and women. In this manuscript, we present a protocol that combines ovarian tissue freezing with the cryopreservation of mature/immature oocytes retrieved from ovarian tissue samples, improving the reproductive potential of fertility preservation. Appropriate collection, handling, and storage of ovarian tissue and oocytes before, during, and after the cryopreservation will be described. The subsequent use and safety of cryopreserved/thawed ovarian tissue samples and oocytes will also be discussed, as well as the optimal timing for in vitro maturation of immature oocytes. We recommend the systematic use of this protocol in fertility preservation of prepubertal girls and women as it increases the whole reproductive potential of fertility preservation (i.e., oocyte vitrification in addition of OTC) and also improves the safety and use of fertility preservation (i.e., thawing of oocytes versus ovarian graft), maximizing the chance of successful childbirth for the patients at risk of POI.

We propose a protocol for fertility preservation in prepubertal girls and women at risk of premature ovarian insufficiency. It combines ovarian tissue freezing and cryopreservation of oocytes retrieved from ovarian tissue. This strategy improves the safety and optimizes the reproductive potential of fertility preservation, maximizing the chance of childbirth.

Human ovarian tissue cryopreservation (OTC) is increasingly used worldwide to preserve female fertility in prepubertal girls and women at risk of ...

Fertility Preservation Through Oocyte Vitrification: Clinical and Laboratory Perspectives

Preserving female fertility is crucial in a multifunctional healthcare system that takes care of patients' future quality of life. Oocyte cryopreservation is recognized by several international scientific societies as the gold standard for fertility preservation in postpubertal women, for both medical and non-medical indications. The main medical indications are oncologic diseases, gynecologic diseases such as severe endometriosis, systemic diseases compromising the ovarian reserve, and genetic conditions involving premature menopause. This paper describes the whole clinical and laboratory work-up of a fertility preservation treatment by outlining recommendations for objective and evidence-based counseling. Furthermore, it focuses on the effectiveness of the procedure and describes the most appropriate strategies to fully exploit the ovarian reserve and maximize the number of oocytes retrieved in the shortest possible time. The evaluation of the ovarian reserve, the definition of an ideal stimulation protocol, as well as oocyte retrieval, denudation, and vitrification procedures have been detailed along with approaches to maximize their efficacy, efficiency, and safety.

Oocyte cryopreservation is recognized by several international scientific societies as the gold standard for fertility preservation in postpubertal women. Appropriate clinical and laboratory strategies ensure maximum efficacy, efficiency, and safety of fertility preservation treatments.

Preserving female fertility is crucial in a multifunctional healthcare system that takes care of patients' future quality of life. Oocyte cryopreservation ...

Laparoscopic Oocyte Retrieval and Cryopreservation during Vaginoplasty for Treatment of Mayer-Rokitansky-Kuster-Hauser Syndrome

In Mayer-Rokitansky-Kuster-Hauser Syndrome (MRKHS) patients who are scheduled for laparoscopic vaginoplasty and have a desire for biological motherhood, we propose that a concomitant laparoscopic oocyte retrieval for cryopreservation is performed. Oocyte retrieval is pursued at the beginning of the laparoscopy. Right and left 5 mm trocars are positioned, through which a 17 G ovum aspiration needle is used for puncture of the right and left ovaries, respectively. To facilitate exposure of the follicles, the ovaries are mobilized and held with laparoscopic forceps.
When aspirating multiple follicles near each other, the needle tip is retained in the ovary to reduce the number of times that the ovarian cortex is transfixed and due to the inherent risk of bleeding. Subsequent steps are unchanged compared to the Davydov laparoscopic modified technique for vaginoplasty. Prior to surgery, controlled ovarian stimulation is performed with a gonadotropin hormone-releasing hormone (Gn-RH) antagonist protocol, and the concomitant procedure of oocyte retrieval and vaginoplasty is scheduled 36 h after the final follicular maturation trigger. Follicular fluid is collected in the same 10 mL sterile tubes used during transvaginal oocyte retrieval and transferred in a warming block (37 &#176;C) to the assisted reproduction laboratory, where mature (metaphase II) oocytes are vitrified.
In this case, a series of 23 women with MRKH, oocytes were successfully retrieved and cryopreserved in all patients; vaginoplasty was subsequently conducted without modifications, and the inpatient and outpatient postoperative care (day of urinary catheter removal, day of hospital discharge, dilator use, and comfort at follow-up) remained unaffected. One postoperative complication occurred in one patient (fever developing on day 5 post surgery and intraperitoneal fluid detection on transabdominal ultrasound) and resolved after conservative treatment. Rather than performing surgical vaginoplasty and delaying oocyte retrieval in MRKH patients, this approach combines both procedures in a single laparoscopy, thereby minimizing surgical invasiveness and anesthesiologic risks.

A dedicated team can offer Mayer-Rokitansky-Kuster-Hauser patients the option to perform controlled ovarian stimulation and oocyte cryopreservation at the time of laparoscopic vaginoplasty.

In Mayer-Rokitansky-Kuster-Hauser Syndrome (MRKHS) patients who are scheduled for laparoscopic vaginoplasty and have a desire for biological motherhood, ...

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.

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 ...

Biology

Follicular Viability Assessment in Vitrified Human Ovarian Cortex Tissues

Vitrification of Ovarian Cortex Tissue to Achieve a Glassy State of Aggregation

Ovarian tissue cryopreservation (OTC) is an important option for fertility preservation. For patients whose gonadotoxic treatments cannot be postponed or for pre-pubertal girls, it is often the only option for fertility protection. Cryopreservation can be performed either by vitrification or by slow freezing. Slow freezing is currently the standard approach. An increasing number of studies indicate that vitrification can replace slow freezing in the state-of-the-art in vitro fertilization (IVF) laboratories, significantly improving thawing survival rates and simplifying the technical aspects of cryopreservation. A metal grid-based, high-throughput protocol for rapid vitrification of ovarian cortex tissue, suitable for clinical routine, is described. The sterilization of metal grids and liquid nitrogen ensures high quality, meeting good manufacturing practice (GMP) standards. Vitrification was conducted to ensure ultra-rapid cooling rates. Instead of slowly thawing, samples were rapidly warmed. To assess follicular viability, calcein staining was performed both prior to cryopreservation and after rapid warming. The successful application of vitrification and rapid warming using metal grids is reported. No significant differences in follicular viability were observed prior to vitrification and after rapid warming. These results substantiate the high capacity of tissue vitrification for clinical routine applications as a potential substitute for the widely used slow-freezing method.

Author Spotlight: Advancing Fertility Preservation in Young Female Cancer Patients Through Ovarian Tissue Vitrification and In Vitro Follicular Growth

A protocol for the vitrification of ovarian tissue, as an alternative cryopreservation method to the widely used slow freezing protocol, is presented.

Ovarian tissue cryopreservation (OTC) is an important option for fertility preservation. For patients whose gonadotoxic treatments cannot be postponed or ...

Improving IVF Outcomes with Partial Granulosa Cell Removal Post-Retrieval

The Immediate Partial Removal of Cumulus-Oocyte Complexes: A Refined Approach for Rapid Observation of In Vitro Fertilization

Despite the rapid advancements in clinical and laboratory technologies for in vitro fertilization (IVF), a significant proportion (10%-15%) of patients continue to experience fertilization disorders, leading to low fertilization rates and the production of nonviable embryos. Short-term fertilization involves the early removal of granulosa cells to observe the extrusion of the second polar body, enabling the assessment of fertilization and early remedial measures to address low fertilization rates and complete fertilization failure. However, the observation of short-term fertilization in IVF is impeded by challenges such as excessively large unprocessed clusters of cumulus-oocyte complexes and adhesion between eggs, necessitating complex external procedures for granulosa cell removal, and prolonged observation duration. To tackle this issue, this study proposes a method of immediate partial removal of granulosa cells post egg retrieval. This approach streamlines subsequent stages of short-term fertilization and traditional fertilization monitoring, reduces the time needed for oocyte extrusion, minimizes the likelihood of external environmental impacts on the oocytes, and decreases the risk of missing the critical fertilization observation window. Consequently, the study yields novel clinical evidence aimed at enhancing the operational efficiency of embryonic laboratories in IVF.

Author Spotlight: Evaluating the Impact of Immediate Partial Removal of Cumulus-Oocyte Complexes on Fertilization Efficiency and Embryo Quality

Here, we present an optimized method for promptly removing a portion of cumulus-oocyte complexes following egg retrieval to expedite the IVF observation process, reducing the time required for granulosa cell removal, minimizing oocyte exposure to external elements, and does not hinder embryo development, ultimately improving the efficiency of IVF laboratory procedures.

Despite the rapid advancements in clinical and laboratory technologies for in vitro fertilization (IVF), a significant proportion (10%-15%) of patients ...

Minimum Volume Vitrification of Immature Feline Oocytes

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