Name: minimum volume vitrification of immature feline oocytes
Uploaded: 2020-06-24T15:00:00
Description: Watch this Scientific Journal Video about Minimum Volume Vitrification of Immature Feline Oocytes at JoVE.com

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 sulph...

<ol>
	<li>Bankowski, B., Lyerly, A., Faden, R., Wallach, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+social+implications+of+embryo+cryopreservation.">The social implications of embryo cryopreservation.</a> Fertility and Sterility. 84 (4), 823-832 (2005).</li><li>Saragusty, J., Arav, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+progress+in+oocyte+and+embryo+cryopreservation+by+slow+freezing+and+vitrification.">Current progress in oocyte and embryo cryopreservation by slow freezing and vitrification.</a> Reproduction. 141 (1), 1-19 (2011).</li><li>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=Gamete+cryopreservation+in+the+domestic+cat.">Gamete cryopreservation in the domestic cat.</a> Theriogenology. 66 (1), 101-111 (2006).</li><li>Thongphakdee, A., Tipkantha, W., Punkong, C., Chatdarong, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+and+controlling+ovarian+activity+in+wild+felids.">Monitoring and controlling ovarian activity in wild felids.</a> Theriogenology. 109, 14-21 (2018).</li><li>Parks, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypothermia+and+mammalian+gametes.">Hypothermia and mammalian gametes.</a> Reproductive Tissue Banking. , 229-261 (1997).</li><li>Carabatsos, M. J., Sellitto, C., Goodenough, D. A., Albertini, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oocyte-granulosa+cell+heterologous+gap+junctions+are+required+for+the+coordination+of+nuclear+and+cytoplasmic+meiotic+competence.">Oocyte-granulosa cell heterologous gap junctions are required for the coordination of nuclear and cytoplasmic meiotic competence.</a> Developmental Biology. 226 (2), 167-179 (2000).</li><li>Murakami, M., 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=Blastocysts+derived+from+in+vitro-fertilized+cat+oocytes+after+vitrification+and+dilution+with+sucrose.">Blastocysts derived from in vitro-fertilized cat oocytes after vitrification and dilution with sucrose.</a> Cryobiology. 48 (3), 341-348 (2004).</li><li>Merlo, B., Iacono, E., Regazzini, M., Zambelli, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cat+blastocysts+produced+in+vitro+from+oocytes+vitrified+using+the+cryoloop+technique+and+cryopreserved+electroejaculated+semen.">Cat blastocysts produced in vitro from oocytes vitrified using the cryoloop technique and cryopreserved electroejaculated semen.</a> Theriogenology. 70 (1), 126-130 (2008).</li><li>Luciano, A. M., 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=Effect+of+different+cryopreservation+protocols+on+cytoskeleton+and+gap+junction+mediated+communication+integrity+in+feline+germinal+vesicle+stage+oocytes.">Effect of different cryopreservation protocols on cytoskeleton and gap junction mediated communication integrity in feline germinal vesicle stage oocytes.</a> Cryobiology. 59 (1), 90-95 (2009).</li><li>Comizzoli, P., Wildt, D. E., Pukazhenthi, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+compaction+of+germinal+vesicle+chromatin+is+beneficial+to+survival+of+vitrified+cat+oocytes.">In vitro compaction of germinal vesicle chromatin is beneficial to survival of vitrified cat oocytes.</a> Reproduction in Domestic Animals. 44, 269-274 (2009).</li><li>Alves, A. E., Kozel, A. 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. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+competence+of+domestic+cat+vitrified+oocytes+in+3D+enriched+culture+conditions.">Developmental competence of domestic cat vitrified oocytes in 3D enriched culture conditions.</a> Animals. 9 (6), 329 (2019).</li><li>Colombo, M., Morselli, M. G., Apparicio, M., 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=Granulosa+cells+in+three-dimensional+culture:+A+follicle-like+structure+for+domestic+cat+vitrified+oocytes.">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/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>

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 ...

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