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>

<ol>
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C., Luvoni, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vitrification+with+DAP+213+and+Cryotop+of+ex+situ+and+in+situ+feline+cumulus-oocyte+complexes.">Vitrification with DAP 213 and Cryotop of ex situ and in situ feline cumulus-oocyte complexes.</a> Reproduction in Domestic Animals. 47 (6), 1003-1008 (2012).</li><li>Kuwayama, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+efficient+vitrification+for+cryopreservation+of+human+oocytes+and+embryos:+The+Cryotop+method.">Highly efficient vitrification for cryopreservation of human oocytes and embryos: The Cryotop method.</a> Theriogenology. 67 (1), 73 (2007).</li><li>Pope, C. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+survival+of+domestic+cat+oocytes+after+vitrification,+intracytoplasmic+sperm+injection+and+embryo+transfer.">In vivo survival of domestic cat oocytes after vitrification, intracytoplasmic sperm injection and embryo transfer.</a> Theriogenology. 77 (3), 531-538 (2012).</li><li>Galiguis, J., G&#243;mez, M. C., Leibo, S. P., Pope, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Birth+of+a+domestic+cat+kitten+produced+by+vitrification+of+lipid+polarized+in+vitro+matured+oocytes.">Birth of a domestic cat kitten produced by vitrification of lipid polarized in vitro matured oocytes.</a> Cryobiology. 68 (3), 459-466 (2014).</li><li>Fernandez-Gonzalez, L., Jewgenow, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryopreservation+of+feline+oocytes+by+vitrification+using+commercial+kits+and+slush+nitrogen+technique.">Cryopreservation of feline oocytes by vitrification using commercial kits and slush nitrogen technique.</a> Reproduction in Domestic Animals. 52, 230-234 (2017).</li><li>Colombo, M., Morselli, M. G., Tavares, M. R., Apparicio, M., Luvoni, G. 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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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.">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., P&#233;rez, S., Ruiz, A., Pellicer, A., Remoh&#237;, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+concomitant+outcome+achieved+with+fresh+and+cryopreserved+donor+oocytes+vitrified+by+the+Cryotop+method.">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><li>Mullen, S. F., Critser, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+science+of+cryobiology.">The science of cryobiology.</a> Oncofertility - Fertility preservation for Cancer Survivors. , 83-109 (2007).</li><li>Fahy, G. M., Wowk, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+of+cryopreservation+by+vitrification.">Principles of cryopreservation by vitrification.</a> Methods in Molecular Biology. 1257, 21-82 (2015).</li><li>Apparicio, M., Ruggeri, E., Luvoni, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vitrification+of+immature+feline+oocytes+with+a+commercial+kit+for+bovine+embryo+vitrification.">Vitrification of immature feline oocytes with a commercial kit for bovine embryo vitrification.</a> Reproduction in Domestic Animals. 48 (2), 240-244 (2013).</li><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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+current+challenges+to+efficient+immature+oocyte+cryopreservation.">The current challenges to efficient immature oocyte cryopreservation.</a> Journal of Assisted Reproduction and Genetics. 30 (12), 1531-1539 (2013).</li><li>Moussa, M., Shu, J., Zhang, X., Zeng, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryopreservation+of+mammalian+oocytes+and+embryos:+current+problems+and+future+perspectives.">Cryopreservation of mammalian oocytes and embryos: current problems and future perspectives.</a> Science China Life Sciences. 57 (9), 903-914 (2014).</li><li>Luvoni, G., Pellizzari, P., Battocchio, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+slow+and+ultrarapid+freezing+on+morphology+and+resumption+of+meiosis+in+immature+cat+oocytes.">Effects of slow and ultrarapid freezing on morphology and resumption of meiosis in immature cat oocytes.</a> Journal of Reproduction and Fertility. Supplement. 51, 93-98 (1997).</li><li>Calvo, J. J., Robert, D., Viqueira, M., Lombide, P., Calvo, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vitrified+and+in+vitro+matured+oocytes+viability+from+adult+housecat+(Felis+catus)+in+reproductive+season.">Vitrified and in vitro matured oocytes viability from adult housecat (Felis catus) in reproductive season.</a> International Journal of Morphology. 33 (4), (2015).</li><li>Nowak, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+viability+of+Serval+(Leptailurus+serval)+and+Pallas+Cat+(Felis+manul)+oocytes+after+cryopreservation+using+the+Rapid-I+Method.">The viability of Serval (Leptailurus serval) and Pallas Cat (Felis manul) oocytes after cryopreservation using the Rapid-I Method.</a> Cryo Letters. 40 (4), 226-230 (2019).</li></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 &#38; tips</td>
			<td>/</td>
			<td>/</td>
			<td>Needed to pipette 20, 100, 240 and 300 &#181;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&#186;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 &#34;Cooling Rack&#34;</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. 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.

Video: Minimum Volume Vitrification of Immature Feline Oocytes

Expression of Fluorescent Proteins in Branchiostoma lanceolatum by mRNA Injection into Unfertilized Oocytes

We report here a robust and efficient protocol for the expression of fluorescent proteins after mRNA injection into unfertilized oocytes of the cephalochordate amphioxus, Branchiostoma lanceolatum. We use constructs for membrane and nuclear targeted mCherry and eGFP that have been modified to accommodate amphioxus codon usage and Kozak consensus sequences. We describe the type of injection needles to be used, the immobilization protocol for the unfertilized oocytes, and the overall injection set-up. This technique generates fluorescently labeled embryos, in which the dynamics of cell behaviors during early development can be analyzed using the latest in vivo imaging strategies. The development of a microinjection technique in this amphioxus species will allow live imaging analyses of cell behaviors in the embryo as well as gene-specific manipulations, including gene overexpression and knockdown. Altogether, this protocol will further consolidate the basal chordate amphioxus as an animal model for addressing questions related to the mechanisms of embryonic development and, more importantly, to their evolution.

Expression of Fluorescent Proteins in Branchiostoma lanceolatum by mRNA Injection into Unfertilized Oocytes

We report here the robust and efficient expression of fluorescent proteins after mRNA injection into unfertilized oocytes of Branchiostoma lanceolatum. The development of the microinjection technique in this basal chordate will pave the way for far-reaching technical innovations in this emerging model system, including in vivo imaging and gene-specific manipulations.

We report here a robust and efficient protocol for the expression of fluorescent proteins after mRNA injection into unfertilized oocytes of the ...

Isolation and Characterization of Mouse Antral Oocytes Based on Nucleolar Chromatin Organization

This protocol describes a simple and quick method to isolate and characterize mouse antral GV (Germinal Vesicle) oocytes as able (SN, Surrounded Nucleolus) or unable (NSN, Not Surrounded Nucleolus) to develop to the blastocyst stage after in vitro maturation (IVM) and in vitro fertilization (IVF). It makes use of Hoeschst33342 (or any other DNA intercalating dye) able to bind to the heterochromatin of the nucleolus showing a ring in the SN oocytes or not, like in the NSN oocytes. This represents the easiest and quickest way to sort both antral oocytes that can be eventually used for IVM or IVF procedures. 
Briefly, the protocol consists of the following steps: hormone injection to stimulate follicular growth; isolation of the oocytes at the GV stage from the antral compartment by puncturing the ovary with a sterile needle; preparation of thin glass pipettes for mouth pipetting of the oocytes; sorting of the oocytes with Hoechst33342 prepared at a supravital concentration; IVM, IVF or any other molecular/cellular analysis.
Unfortunately there are still few evidences to sort SN and NSN oocytes using less invasive techniques. If and once they will be identified, they could be potentially applied to human assisted reproductive technologies, although with several aspects that should be modified. To date, this technique has potential implications to dramatically increase IVM and IVF successful procedures in both endangered and species with economic interest.

Here we present a protocol for the characterization of mouse antral oocytes based on nucleolar organization.

This protocol describes a simple and quick method to isolate and characterize mouse antral GV (Germinal Vesicle) oocytes as able (SN, Surrounded ...

Modified MicroSecure Vitrification: A Safe, Simple and Highly Effective Cryopreservation Procedure for Human Blastocysts

Clinical embryo vitrification evolved with the development of unique vitrification devices in the 21st century and with the misconception that ultra-rapid cooling in an &#34;open&#34; system (i.e., direct LN2 contact) was a necessity to optimize vitrification success. The dogma surrounding the importance of cooling rates led to unsafe practices subject to technical variation and to the creation of vitrification devices that disregarded important quality-control factors (e.g., ease of use, repeatability, reliability, labeling security, and storage safety). Understanding the quality-control flaws of other devices allowed for the development of a safe, secure, repeatable, and reliable &#181;S-VTF method aimed to minimize intra- and inter-technician variation. Equally important, it combined the availability of two existing FDA-compliant devices: 1) a 0.3-mL ionomeric resin embryo straw with internalized, dual-colored, tamper-proof labeling with repeatable weld seal potential; and 2) shortened, commonly-used, 300-&#181;m ID sterile flexipettes to directly load the embryo(s) in order to create a highly-effective global vitrification device. Like other aseptic, closed vitrification systems (e.g., High Security Vitrification (HSV), Rapid-i, and VitriSafe) effectively used in reproductive medicine, microSecure Vitrification (&#181;S-VTF) has proven that it can achieve high post-warming survival and pregnancy outcomes with its attention to simplicity, and reduced technical variation. Although the 0.3-mL embryo straw containing an internal hydrophobic plug was commercially replaced with a standard semen straw possessing cotton-polyvinyl pyrrolidone (PVP) plugs, it maintained its ionomeric resin composition to ensure weld sealing. However, the cotton plugs can wick out the fluid-embryo contents of the flexipettes upon contact. A modified &#181;S-VTF method was adapted to include an additional internal weld seal before the plug on the device loading side. The added technical step to the &#181;S-VTF procedure has not affected its successful application, as high survival rates (&#62; 95%) and pregnancy rates continue today.

MicroSecure Vitrification was developed as a non-commercial, aseptic closed vitrification device system that is compliant with FDA good manufacturing and tissue-handling practices. Due to the withdrawal of hydrophobic plugged embryo straws from the industry, the vitrification procedure was modified to include an inner seal before the standard internal cotton plug.

Clinical embryo vitrification evolved with the development of unique vitrification devices in the 21st century and with the misconception that ultra-rapid ...

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 ...

Generation of Genetically Modified Mice through the Microinjection of Oocytes

The use of genetically modified mice has significantly contributed to studies on both physiological and pathological in vivo processes. The pronuclear injection of DNA expression constructs into fertilized oocytes remains the most commonly used technique to generate transgenic mice for overexpression. With the introduction of CRISPR technology for gene targeting, pronuclear injection into fertilized oocytes has been extended to the generation of both knockout and knockin mice. This work describes the preparation of DNA for injection and the generation of CRISPR guides for gene targeting, with a particular emphasis on quality control. The genotyping procedures required for the identification of potential founders are critical. Innovative genotyping strategies that take advantage of the "multiplexing" capabilities of CRISPR are presented herein. Surgical procedures are also outlined. Together, the steps of the protocol will allow for the generation of genetically modified mice and for the subsequent establishment of mouse colonies for a plethora of research fields, including immunology, neuroscience, cancer, physiology, development, and others.

The microinjection of mouse oocytes is commonly used for both classic transgenesis (i.e., the random integration of transgenes) and CRISPR-mediated gene targeting. This protocol reviews the latest developments in microinjection, with a particular emphasis on quality control and genotyping strategies.

The use of genetically modified mice has significantly contributed to studies on both physiological and pathological in vivo processes. The pronuclear ...

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

Infertility clinics would benefit from the ability to select developmentally competent vs. incompetent oocytes using non-invasive procedures, thus improving the overall pregnancy outcome. We recently developed a classification method based on microscopic live observations of mouse oocytes during their in vitro maturation from the germinal vesicle (GV) to the metaphase II stage, followed by the analysis of the cytoplasmic movements occurring during this time-lapse period. Here, we present detailed protocols of this procedure. Oocytes are isolated from fully-grown antral follicles and cultured for 15 h inside a microscope equipped for time-lapse analysis at 37 &#176;C and 5% CO2. Pictures are taken at 8 min intervals. The images are analyzed using the Particle Image Velocimetry (PIV) method that calculates, for each oocyte, the profile of Cytoplasmic Movement Velocities (CMVs) occurring throughout the culture period. Finally, the CMVs of each single oocyte are fed through a mathematical classification tool (Feed-forward Artificial Neural Network, FANN), which predicts the probability of a gamete to be developmentally competent or incompetent with an accuracy of 91.03%. This protocol, set up for the mouse, could now be tested on oocytes of other species, including humans.

Here, we present a protocol for non-invasive assessment of oocyte developmental competence performed during their in vitro maturation from the germinal vesicle to the metaphase II stage. This method combines time-lapse imaging with particle image velocimetry (PIV) and neural network analyses.

Infertility clinics would benefit from the ability to select developmentally competent vs. incompetent oocytes using non-invasive procedures, thus ...

Minimally Invasive Embryo Transfer and Embryo Vitrification at the Optimal Embryo Stage in Rabbit Model

Assisted reproductive techniques (ARTs), such as in vitro embryo culture or embryo cryopreservation, affect natural development patterns with perinatal and postnatal consequences. To ensure the innocuousness of ART applications, studies in animal models are necessary. In addition, as a last step, embryo development studies require evaluation of their capacity to develop full-term healthy offspring. Here, embryo transfer to the uterus is indispensable to perform any ARTs-related experiment.
The rabbit has been used as a model organism to study mammalian reproduction for over a century. In addition to its phylogenetic proximity to the human species and its small size and low maintenance cost, it has important reproductive characteristics such as induced ovulation, a chronology of early embryonic development similar to humans and a short gestation that allow us to study the consequences of ART application easily. Moreover, ARTs (such as intracytoplasmic sperm injection, embryo culture, or cryopreservation) are applied with suitable efficiency in this species.
Using the laparoscopic embryo transfer technique and the cryopreservation protocol presented in this article, we describe 1) how to transfer embryos through an easy, minimally invasive technique and 2) an effective protocol for long-term storage of rabbit embryos to provide time-flexible logistical capacities and the ability to transport the sample. The outcomes obtained after transferring rabbit embryos at different developmental stages indicate that morula is the ideal stage for rabbit embryo recovery and transfer. Thus, an oviductal embryo transfer is required, justifying the surgical procedure. Furthermore, rabbit morulae are successfully vitrified and laparoscopically transferred, proving the effectiveness of the described techniques.

Assisted reproductive techniques (ARTs) are in continuous evaluation to improve outcomes and reduce the associated risks. This manuscript describes a minimally invasive embryo transfer procedure with an efficient cryopreservation protocol that allows the use of rabbits as an ideal animal model of human reproduction.

Assisted reproductive techniques (ARTs), such as in vitro embryo culture or embryo cryopreservation, affect natural development patterns with perinatal ...

Human Blastocyst Biopsy and Vitrification

Blastocyst biopsy is performed to obtain a reliable genetic diagnosis during IVF cycles with preimplantation genetic testing. Then, the ideal workflow entails a safe and efficient vitrification protocol, due to the turnaround time of the diagnostic techniques and to transfer the selected embryo(s) on a physiological endometrium in a following natural cycle. A biopsy approach encompassing the sequential opening of the zona pellucida and retrieval of 5-10 trophectoderm cells (ideally 7-8) limits both the number of manipulations required and the exposure of the embryo to sub-optimal environmental conditions. After proper training, the technique was reproducible across different operators in terms of timing of biopsy (~8 min, ranging 3-22 min based on the number of embryos to biopsy per dish), conclusive diagnoses obtained (~97.5%) and live birth rates after vitrified-warmed euploid blastocyst transfer (&#62;40%). The survival rate after biopsy, vitrification and warming was as high as 99.8%. The re-expansion rate at 1.5 h from warming was as high as 97%, largely dependent on the timing between biopsy and vitrification (ideally &#8804;30 min), blastocyst morphological quality and day of biopsy. In general, it is better to vitrify a collapsed blastocyst; therefore, in non-PGT cycles, laser-assisted artificial shrinkage might be performed to induce embryo collapse prior to cryopreservation. The most promising future perspective is the non-invasive analysis of the IVF culture media after blastocyst culture as a putative source of embryonic DNA. However, this potential avant-garde is still under investigation and a reliable protocol yet needs to be defined and validated.

Blastocyst biopsy and vitrification are required to efficiently conduct preimplantation genetic testing. An approach entailing the sequential opening of the zona pellucida and retrieval of 7-8 trophectoderm cells in day 5-7 post-insemination limits both the number of manipulations required and the exposure of the embryo to sub-optimal environmental conditions.

Blastocyst biopsy is performed to obtain a reliable genetic diagnosis during IVF cycles with preimplantation genetic testing. Then, the ideal workflow ...

In Vitro Culture Strategy for Oocytes from Early Antral Follicle in Cattle

The limited reserve of mature, fertilizable oocytes represents a major barrier for the success of assisted reproduction in mammals. Considering that during the reproductive life span only about 1% of the oocytes in an ovary mature and ovulate, several techniques have been developed to increase the exploitation of the ovarian reserve to the growing population of non-ovulatory follicles. Such technologies have allowed interventions of fertility preservation, selection programs in livestock, and conservation of endangered species. However, the vast potential of the ovarian reserve is still largely unexploited. In cows, for instance, some attempts have been made to support in vitro culture of oocytes at specific developmental stages, but efficient and reliable protocols have not yet been developed. Here we describe a culture system that reproduce the physiological conditions of the corresponding follicular stage, defined to develop in vitro growing oocytes collected from bovine early antral follicles to the fully-grown stage, corresponding to the medium antral follicle in vivo. A combination of hormones and a phosphodiesterase 3 inhibitor was used to prevent untimely meiotic resumption and to guide oocyte&#39;s differentiation.

We describe the procedures for isolation of growing oocytes from ovarian follicles at early stages of development, as well as the setup of an in vitro culture system which can support the growth and differentiation up to the fully-grown stage. &#160;

The limited reserve of mature, fertilizable oocytes represents a major barrier for the success of assisted reproduction in mammals. Considering that ...

Separation of Follicular Cells and Oocytes in Ovarian Follicles of Zebrafish

Zebrafish has become an ideal model to study the ovarian development of vertebrates. The follicle is the basic unit of the ovary, which consists of oocytes and surrounding follicular cells. It is vital to separate both follicular cells and oocytes for various research purposes such as for primary culture of follicular cells, analysis of gene expression, oocyte maturation and in vitro fertilization, etc. The conventional method uses forceps to separate both compartments, which is laborious, time consuming and has high damage to the oocyte. Here, we have established a simple method to separate both compartments using a pulled glass capillary. Under a stereomicroscope, oocytes and follicular cells can be easily separated by pipetting in a pulled fine glass capillary (the diameter depends on the follicle diameter). Compared with the conventional method, this new method has high efficiency in separating both oocytes and follicular cells and has low damage to the oocytes. More importantly, this method can be applied to early-stage follicles including at the pre-vitellogenesis stage. Thus, this simple method can be used to separate follicular cells and oocytes of zebrafish.

Here, we present a simple method for separating follicular cells and oocytes in zebrafish ovarian follicles, which will facilitate investigations of ovarian development in zebrafish.

Zebrafish has become an ideal model to study the ovarian development of vertebrates. The follicle is the basic unit of the ovary, which consists of ...