The authors received no specific funding for this work. Professor Maria Grazia Cappai and Dr. Valeria Pasciu are gratefully acknowledged for the video voiceover and for setting up the lab during the video making.

The animal protocol and the implemented procedures described below are in accordance with the ethical guidelines in force at the University of Sassari, in compliance with the European Union Directive 86/609/EC and the recommendation of the Commission of the European Communities 2007/526/EC.
1. Preparation of media for oocyte manipulation
<ol>
	<li>Prepare the medium for transport of collected ovaries by supplementing Dulbecco&#39;s phosphate buffered saline with 0.1 g/L penicillin and 0.1 g/...

<ol>
	<li>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=Cryopreservation+of+oocytes+and+embryos.">Cryopreservation of oocytes and embryos.</a> Theriogenology. 81 (1), 96-102 (2014).</li><li>Mullen, S. F., Fahy, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+chronologic+review+of+mature+oocyte+vitrification+research+in+cattle,+pigs,+and+sheep.">A chronologic review of mature oocyte vitrification research in cattle, pigs, and sheep.</a> Theriogenology. 78 (8), 1709-1719 (2012).</li><li>Hwang, I. S., Hochi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+progress+in+cryopreservation+of+bovine+oocytes.">Recent progress in cryopreservation of bovine oocytes.</a> BioMed Research International. 2014, (2014).</li><li>Berlinguer, F., 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=Effects+of+trehalose+co-incubation+on+in+vitro+matured+prepubertal+ovine+oocyte+vitrification.">Effects of trehalose co-incubation on in vitro matured prepubertal ovine oocyte vitrification.</a> Cryobiology. 55 (1), (2007).</li><li>Quan, G., Wu, G., Hong, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oocyte+Cryopreservation+Based+in+Sheep:+The+Current+Status+and+Future+Perspective.">Oocyte Cryopreservation Based in Sheep: The Current Status and Future Perspective.</a> Biopreservation and Biobanking. 15 (6), 535-547 (2017).</li><li>Succu, S., 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=A+recovery+time+after+warming+restores+mitochondrial+function+and+improves+developmental+competence+of+vitrified+ovine+oocytes.">A recovery time after warming restores mitochondrial function and improves developmental competence of vitrified ovine oocytes.</a> Theriogenology. 110, (2018).</li><li>Succu, S., 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=Vitrification+of+in+vitro+matured+ovine+oocytes+affects+in+vitro+pre-implantation+development+and+mRNA+abundance.">Vitrification of in vitro matured ovine oocytes affects in vitro pre-implantation development and mRNA abundance.</a> Molecular Reproduction and Development. 75 (3), (2008).</li><li>Succu, S., 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=Vitrification+Devices+Affect+Structural+and+Molecular+Status+of+In+Vitro+Matured+Ovine+Oocytes.">Vitrification Devices Affect Structural and Molecular Status of In Vitro Matured Ovine Oocytes.</a> Molecular Reproduction and Development. 74, 1337-1344 (2007).</li><li>Hosseini, S. M., Asgari, V., Hajian, M., Nasr-Esfahani, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytoplasmic,+rather+than+nuclear-DNA,+insufficiencies+as+the+major+cause+of+poor+competence+of+vitrified+oocytes.">Cytoplasmic, rather than nuclear-DNA, insufficiencies as the major cause of poor competence of vitrified oocytes.</a> Reproductive BioMedicine Online. , (2015).</li><li>Succu, S., 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=Calcium+concentration+in+vitrification+medium+affects+the+developmental+competence+of+in+vitro+matured+ovine+oocytes.">Calcium concentration in vitrification medium affects the developmental competence of in vitro matured ovine oocytes.</a> Theriogenology. 75 (4), (2011).</li><li>Sanaei, B., 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=An+improved+method+for+vitrification+of+in+vitro+matured+ovine+oocytes;+beneficial+effects+of+Ethylene+Glycol+Tetraacetic+acid,+an+intracellular+calcium+chelator.">An improved method for vitrification of in vitro matured ovine oocytes; beneficial effects of Ethylene Glycol Tetraacetic acid, an intracellular calcium chelator.</a> Cryobiology. 84, 82-90 (2018).</li><li>Quan, G. B., Wu, G. Q., Wang, Y. J., Ma, Y., Lv, C. R., Hong, Q. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Meiotic+maturation+and+developmental+capability+of+ovine+oocytes+at+germinal+vesicle+stage+following+vitrification+using+different+cryodevices.">Meiotic maturation and developmental capability of ovine oocytes at germinal vesicle stage following vitrification using different cryodevices.</a> Cryobiology. 72 (1), 33-40 (2016).</li><li>Fern&#225;ndez-Reyez, F., 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=maturation+and+embryo+development+in+vitro+of+immature+porcine+and+ovine+oocytes+vitrified+in+different+devices.">maturation and embryo development in vitro of immature porcine and ovine oocytes vitrified in different devices.</a> Cryobiology. 64 (3), 261-266 (2012).</li><li>Ahmadi, E., Shirazi, A., Shams-Esfandabadi, N., Nazari, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antioxidants+and+glycine+can+improve+the+developmental+competence+of+vitrified/warmed+ovine+immature+oocytes.">Antioxidants and glycine can improve the developmental competence of vitrified/warmed ovine immature oocytes.</a> Reproduction in Domestic Animals. 54 (3), 595-603 (2019).</li><li>Barrera, N., 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=Impact+of+delipidated+estrous+sheep+serum+supplementation+on+in+vitro+maturation,+cryotolerance+and+endoplasmic+reticulum+stress+gene+expression+of+sheep+oocytes.">Impact of delipidated estrous sheep serum supplementation on in vitro maturation, cryotolerance and endoplasmic reticulum stress gene expression of sheep oocytes.</a> PLoS ONE. 13 (6), (2018).</li><li>Walker, S. K., Hill, J. L., Kleemann, D. O., Nancarrow, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+Ovine+Embryos+in+Synthetic+Oviductal+Fluid+Containing+Amino+Acids+at+Oviductal+Fluid+Concentrations.">Development of Ovine Embryos in Synthetic Oviductal Fluid Containing Amino Acids at Oviductal Fluid Concentrations.</a> Biology of Reproduction. 55 (3), 703-708 (1996).</li><li>Kuwayama, M., Vajta, G., Kato, O., Leibo, S. P. <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+method+for+cryopreservation+of+human+oocytes.">Highly efficient vitrification method for cryopreservation of human oocytes.</a> Reproductive BioMedicine Online. 11 (3), 300-308 (2005).</li><li>Wu, X., Jin, X., Wang, Y., Mei, Q., Li, J., Shi, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+and+spectral+properties+of+novel+chlorinated+pH+fluorescent+probes.">Synthesis and spectral properties of novel chlorinated pH fluorescent probes.</a> Journal of Luminescence. 131 (4), 776-780 (2011).</li><li>Dell'Aquila, M. 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=Prooxidant+effects+of+verbascoside,+a+bioactive+compound+from+olive+oil+mill+wastewater,+on+in+vitro+developmental+potential+of+ovine+prepubertal+oocytes+and+bioenergetic/oxidative+stress+parameters+of+fresh+and+vitrified+oocytes.">Prooxidant effects of verbascoside, a bioactive compound from olive oil mill wastewater, on in vitro developmental potential of ovine prepubertal oocytes and bioenergetic/oxidative stress parameters of fresh and vitrified oocytes.</a> BioMed Research International. 2014, (2014).</li><li>Gadau, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphological+and+quantitative+analysis+on+&#945;-tubulin+modifications+in+glioblastoma+cells.">Morphological and quantitative analysis on &#945;-tubulin modifications in glioblastoma cells.</a> Neuroscience Letters. 687, 111-118 (2018).</li><li>los Reyes, M. D., Palomino, J., Parraguez, V. H., Hidalgo, M., Saffie, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+distribution+and+meiotic+progression+in+canine+oocytes+during+in+vivo+and+in+vitro+maturation.">Mitochondrial distribution and meiotic progression in canine oocytes during in vivo and in vitro maturation.</a> Theriogenology. , (2011).</li><li>Leoni, G. G., 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=Differences+in+the+kinetic+of+the+first+meiotic+division+and+in+active+mitochondrial+distribution+between+prepubertal+and+adult+oocytes+mirror+differences+in+their+developmental+competence+in+a+sheep+model.">Differences in the kinetic of the first meiotic division and in active mitochondrial distribution between prepubertal and adult oocytes mirror differences in their developmental competence in a sheep model.</a> PLoS ONE. 10 (4), (2015).</li><li>Berlinguer, F., 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=Effects+of+trehalose+co-incubation+on+in+vitro+matured+prepubertal+ovine+oocyte+vitrification.">Effects of trehalose co-incubation on in vitro matured prepubertal ovine oocyte vitrification.</a> Cryobiology. 55 (1), 27-34 (2007).</li><li>Serra, E., Gadau, S. D., Berlinguer, F., Naitana, S., Succu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphological+features+and+microtubular+changes+in+vitrified+ovine+oocytes.">Morphological features and microtubular changes in vitrified ovine oocytes.</a> Theriogenology. 148, 216-224 (2020).</li><li>Asgari, V., Hosseini, S. M., Ostadhosseini, S., Hajian, M., Nasr-Esfahani, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time+dependent+effect+of+post+warming+interval+on+microtubule+organization,+meiotic+status,+and+parthenogenetic+activation+of+vitrified+in+vitro+matured+sheep+oocytes.">Time dependent effect of post warming interval on microtubule organization, meiotic status, and parthenogenetic activation of vitrified in vitro matured sheep oocytes.</a> Theriogenology. 75 (5), 904-910 (2011).</li><li>Ciotti, P. 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=Meiotic+spindle+recovery+is+faster+in+vitrification+of+human+oocytes+compared+to+slow+freezing.">Meiotic spindle recovery is faster in vitrification of human oocytes compared to slow freezing.</a> Fertility and Sterility. 91 (6), 2399-2407 (2009).</li><li>Ledda, S., Bogliolo, L., Leoni, G., Naitana, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+Coupling+and+Maturation-Promoting+Factor+Activity+in+In+Vitro-Matured+Prepubertal+and+Adult+Sheep+Oocytes1.">Cell Coupling and Maturation-Promoting Factor Activity in In Vitro-Matured Prepubertal and Adult Sheep Oocytes1.</a> Biology of Reproduction. 65 (1), 247-252 (2001).</li><li>Palmerini, M. G., 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+vitro+maturation+is+slowed+in+prepubertal+lamb+oocytes:+ultrastructural+evidences.">In vitro maturation is slowed in prepubertal lamb oocytes: ultrastructural evidences.</a> Reproductive Biology and Endocrinology. 12, (2014).</li><li>Leoni, G. G., 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=Relations+between+relative+mRNA+abundance+and+developmental+competence+of+ovine+oocytes.">Relations between relative mRNA abundance and developmental competence of ovine oocytes.</a> Molecular Reproduction and Development. 74 (2), 249-257 (2007).</li><li>Succu, S., 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+vitrification+solutions+and+cooling+upon+in+vitro+matured+prepubertal+ovine+oocytes.">Effect of vitrification solutions and cooling upon in vitro matured prepubertal ovine oocytes.</a> Theriogenology. 68 (1), 107-114 (2007).</li><li>Larman, M. G., Sheehan, C. B., Gardner, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium-free+vitrification+reduces+cryoprotectant-induced+zona+pellucida+hardening+and+increases+fertilization+rates+in+mouse+oocytes.">Calcium-free vitrification reduces cryoprotectant-induced zona pellucida hardening and increases fertilization rates in mouse oocytes.</a> Reproduction. 131 (1), 53-61 (2006).</li><li>Yeste, M., Jones, C., Amdani, S. N., Patel, S., Coward, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oocyte+activation+deficiency:+a+role+for+an+oocyte+contribution.">Oocyte activation deficiency: a role for an oocyte contribution.</a> Human Reproduction Update. 22 (1), 23-47 (2016).</li><li>Rienzi, L., 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=Oocyte,+embryo+and+blastocyst+cryopreservation+in+ART:+systematic+review+and+meta-analysis+comparing+slow-freezing+versus+vitrification+to+produce+evidence+for+the+development+of+global+guidance.">Oocyte, embryo and blastocyst cryopreservation in ART: systematic review and meta-analysis comparing slow-freezing versus vitrification to produce evidence for the development of global guidance.</a> Human Reproduction Update. 23 (2), 139-155 (2017).</li><li>De Santis, L., 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=Oocyte+vitrification:+influence+of+operator+and+learning+time+on+survival+and+development+parameters.">Oocyte vitrification: influence of operator and learning time on survival and development parameters.</a> Placenta. 32, 280-281 (2011).</li><li>Zhang, X., Catalano, P. N., Gurkan, U. A., Khimji, I., Demirci, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emerging+technologies+in+medical+applications+of+minimum+volume+vitrification.">Emerging technologies in medical applications of minimum volume vitrification.</a> Nanomedicine. 6 (6), 1115-1129 (2011).</li></ol>

The authors declare they have no competing financial interests.

Oocyte cryopreservation in domestic animals can allow not only the long-term conservation of female genetic resources, but also advance the development of embryonic biotechnologies. Thus, the development of a standard method for oocyte vitrification would advantage both the livestock and the research sector. In this protocol, a complete method for adult sheep oocyte vitrification is presented and could represent a solid starting point for the development of an efficient vitrification system for juvenile oocyte.
<p cl...

Long-term storage of the female gametes can offer a wide range of applications, such as improving domestic animal breeding by genetic selection programs, contributing to preserve biodiversity through the ex-situ wildlife species conservation program, and boosting in vitro biotechnology research and applications thanks to the availability of stored oocytes to be incorporated in in vitro embryo production or nuclear transplantation programs1,2,3. Juvenile oocyte vitrification would also increase genetic gain by shortening the generation interval in breeding programs4. Vitrification by ultra-rapid cooling and warming of oocytes is currently considered a standard approach for livestock oocytes cryopreservation5. In ruminants, before vitrification, oocytes are usually matured in vitro, after retrieval from follicles obtained from abattoir-derived ovaries2. Adult, and especially prepubertal ovaries4,6, can indeed supply a virtually unlimited number of oocytes to be cryopreserved.
In cattle, after oocyte vitrification and warming, blastocyst yields at &#62;10% have been commonly reported by several laboratories during the last decade3. However, in small ruminants oocyte vitrification is still considered relatively new for both juvenile and adult oocytes, and a standard method for sheep oocyte vitrification remains to be established2,5. Despite recent advancements, the vitrified and warmed oocyte indeed presents several functional and structural alterations that limit their developmental potential7,8,9. Thus, few articles have reported blastocyst development at 10% or more in vitrified/warmed sheep oocytes2. Several approaches have been investigated to reduce the above-mentioned alterations: optimizing the composition of the vitrification and thawing solutions10,11; experimenting with the use of different cryo-devices8,12,13; and applying specific treatments during in vitro maturation (IVM)4,14,15 and/or during the recovery time after warming6.
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 culture period.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2&#8242;,7&#8242;-Dichlorofluorescin diacetate</td>
			<td>Sigma-Aldrich</td>
			<td>D-6883</td>
			<td></td>
		</tr>
		<tr>
			<td>Albumin bovine fraction V, protease free</td>
			<td>Sigma-Aldrich</td>
			<td>A3059</td>
			<td></td>
		</tr>
		<tr>
			<td>Bisbenzimide H 33342 trihydrochloride (Hoechst 33342)</td>
			<td>Sigma-Aldrich</td>
			<td>14533</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium chloride (CaCl2 2H20)</td>
			<td>Sigma-Aldrich</td>
			<td>C8106</td>
			<td></td>
		</tr>
		<tr>
			<td>Citric acid</td>
			<td>Sigma-Aldrich</td>
			<td>C2404</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal laser scanning microscope</td>
			<td>Leica Microsystems GmbH,Wetzlar</td>
			<td>TCS SP5 DMI 6000CS</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryotop Kitazato</td>
			<td>Medical Biological Technologies</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cysteamine</td>
			<td>Sigma-Aldrich</td>
			<td>M9768</td>
			<td></td>
		</tr>
		<tr>
			<td>D- (-) Fructose</td>
			<td>Sigma-Aldrich</td>
			<td>F0127</td>
			<td></td>
		</tr>
		<tr>
			<td>D(+)Trehalose dehydrate</td>
			<td>Sigma-Aldrich</td>
			<td>T0167</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Sigma-Aldrich</td>
			<td>D2438</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco Phosphate Buffered Saline</td>
			<td>Sigma-Aldrich</td>
			<td>D8537</td>
			<td></td>
		</tr>
		<tr>
			<td>Egg yolk</td>
			<td>Sigma-Aldrich</td>
			<td>P3556</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylene glycol (EG)</td>
			<td>Sigma-Aldrich</td>
			<td>324558</td>
			<td></td>
		</tr>
		<tr>
			<td>FSH</td>
			<td>Sigma-Aldrich</td>
			<td>F4021</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamic Acid</td>
			<td>Sigma-Aldrich</td>
			<td>G5638</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutaraldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>G5882</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma-Aldrich</td>
			<td>G5516</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Sigma-Aldrich</td>
			<td>G8790</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Sigma-Aldrich</td>
			<td>H4149</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H4034</td>
			<td></td>
		</tr>
		<tr>
			<td>Hypoutarine</td>
			<td>Sigma-Aldrich</td>
			<td>H1384</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted microscope</td>
			<td>Diaphot, Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>L-Alanine</td>
			<td>Sigma-Aldrich</td>
			<td>A3534</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Arginine</td>
			<td>Sigma-Aldrich</td>
			<td>A3784</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Asparagine</td>
			<td>Sigma-Aldrich</td>
			<td>A4284</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Aspartic Acid</td>
			<td>Sigma-Aldrich</td>
			<td>A4534</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Cysteine</td>
			<td>Sigma-Aldrich</td>
			<td>C7352</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Cystine</td>
			<td>Sigma-Aldrich</td>
			<td>C8786</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Sigma-Aldrich</td>
			<td>G3126</td>
			<td></td>
		</tr>
		<tr>
			<td>LH</td>
			<td>Sigma-Aldrich</td>
			<td>L6420</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Histidine</td>
			<td>Sigma-Aldrich</td>
			<td>H9511</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Isoleucine</td>
			<td>Sigma-Aldrich</td>
			<td>I7383</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Leucine</td>
			<td>Sigma-Aldrich</td>
			<td>L1512</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Lysine</td>
			<td>Sigma-Aldrich</td>
			<td>L1137</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Methionine</td>
			<td>Sigma-Aldrich</td>
			<td>M2893</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Ornithine</td>
			<td>Sigma-Aldrich</td>
			<td>O6503</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Phenylalanine</td>
			<td>Sigma-Aldrich</td>
			<td>P5030</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Proline</td>
			<td>Sigma-Aldrich</td>
			<td>P4655</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Serine</td>
			<td>Sigma-Aldrich</td>
			<td>S5511</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Tyrosine</td>
			<td>Sigma-Aldrich</td>
			<td>T1020</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Valine</td>
			<td>Sigma-Aldrich</td>
			<td>V6504</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride heptahydrate (MgSO4.7H2O)</td>
			<td>Sigma-Aldrich</td>
			<td>M2393</td>
			<td></td>
		</tr>
		<tr>
			<td>Makler Counting Chamber</td>
			<td>Sefi-Medical Instruments ltd.Biosigma S.r.l.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Medium 199</td>
			<td>Sigma-Aldrich</td>
			<td>M5017</td>
			<td></td>
		</tr>
		<tr>
			<td>Mineral oil</td>
			<td>Sigma-Aldrich</td>
			<td>M8410</td>
			<td></td>
		</tr>
		<tr>
			<td>MitoTracker Red CM-H2XRos</td>
			<td>ThermoFisher</td>
			<td>M7512</td>
			<td></td>
		</tr>
		<tr>
			<td>New born calf serum heat inactivated (FCS)</td>
			<td>Sigma-Aldrich</td>
			<td>N4762</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin G sodium salt</td>
			<td>Sigma-Aldrich</td>
			<td>P3032</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenol Red</td>
			<td>Sigma-Aldrich</td>
			<td>P3532</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyvinyl alcohol (87-90% hydrolyzed, average mol wt 30,000-70,000)</td>
			<td>Sigma-Aldrich</td>
			<td>P8136</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Chloride (KCl)</td>
			<td>Sigma-Aldrich</td>
			<td>P5405</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium phosphate monobasic (KH2PO4)</td>
			<td>Sigma-Aldrich</td>
			<td>P5655</td>
			<td></td>
		</tr>
		<tr>
			<td>Propidium iodide</td>
			<td>Sigma-Aldrich</td>
			<td>P4170</td>
			<td></td>
		</tr>
		<tr>
			<td>Sheep serum</td>
			<td>Sigma-Aldrich</td>
			<td>S2263</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium azide</td>
			<td>Sigma-Aldrich</td>
			<td>S2202</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium bicarbonate (NaHCO3)</td>
			<td>Sigma-Aldrich</td>
			<td>S5761</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride (NaCl)</td>
			<td>Sigma-Aldrich</td>
			<td>S9888</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium dl-lactate solution syrup</td>
			<td>Sigma-Aldrich</td>
			<td>L4263</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pyruvate</td>
			<td>Sigma-Aldrich</td>
			<td>P2256</td>
			<td></td>
		</tr>
		<tr>
			<td>Sperm Class Analyzer</td>
			<td>Microptic S.L.</td>
			<td>S.C.A. v 3.2.0</td>
			<td></td>
		</tr>
		<tr>
			<td>Statistical software Minitab 18.1</td>
			<td>2017 Minitab</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Stereo microscope</td>
			<td>Olimpus</td>
			<td>SZ61</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptomycin sulfate</td>
			<td>Sigma-Aldrich</td>
			<td>S9137</td>
			<td></td>
		</tr>
		<tr>
			<td>Taurine</td>
			<td>Sigma-Aldrich</td>
			<td>T7146</td>
			<td></td>
		</tr>
		<tr>
			<td>TRIS</td>
			<td>Sigma-Aldrich</td>
			<td>15,456-3</td>
			<td></td>
		</tr>
	</tbody>
</table>

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 cryotolerance of oocyte from juvenile donors is lower compared to adult ones. The first effect observed is a lower post-warming survival rate compared to adult oocytes (Figure 1A; &#967;2 test P&#60;0.001). Juvenile oocytes showed a lower membrane integrity after warming (Figure 1B). The use of trehalose in the maturation medium was intended to verify whether this sugar could reduce cryoinjuries in juvenile oocytes. The data have demonstrated<sup ...

Watch this Scientific Journal Video about Vitrification of In Vitro Matured Oocytes Collected from Adult and Prepubertal Ovaries in Sheep at JoVE.com

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

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 ...

The maintaining of oocyte potential viability after longterm storage represent a tool of great opportunity, as it would improve domestic animal breeding by genetic selection programs, contribute to preserve biodiversity through wildlife species conservation, and increase the research in biotechnology. Juvenile oocyte vitrification was a lot shorter in the generation interval in breeding programs. Vitrifaction by ultrarapid cooling and warming is considered a standard approach in cattle oocyte preservation.However, in sheep this procedure is still considered relatively new and a standard method is lacking. In this video, we describe a protocol for the vitrification of sheep oocyte, collected from both juvenile and adult donors, and in vitro matured to prior to cryopreservation. The protocol include all the procedures from in vitro maturation, vitrification, warming, and post-warming culture.After recovering the Cumulus oocytes complex, where matured in vitro for 24 hours in static a condition that include for juvenile oocyte, the supplementation with hundred micromolar of trehalose in maturation medium. Following in vitro maturation, the oocytes were denuded of Cumulus cells mechanically by gently pipetting and examined under stereo microscope, with 60x magnification. The selection of the oocytes to submit at the vitrification procedures represent the first important step to warranty the successful application of this procedure.Only those with the uniform cytoplasm, with homogeneous distribution of lipid droplets, integrity of the all Emma wide and continuous pivotal in space, compact zona pellucida the outer diameter of about 19 micrometers and showing the extrusion of the first polar body, and thus at the M2, stage must be selected. Vitrification was performed using the minimum essential volume method, with a Cryotops device. After selection, a group of five juvenile oocytes were conditioned at 38.5 Celsius degrees in base medium for two minutes.The oocytes were then dehydrated by three minutes exposure to a calibration solution. The loading of the oocyte in the vitrification device used is an important and critical step. Cryotop uses a polypropylene strip attached to a holder.In this method, the oocytes in the vitrification solution, less than 0 point milliliter are rapidly loaded with a glass capillary on top of the film strip. Then the solution must be removed, leaving behind a thin layer enough to cover the cells to be cryopreserved. Before being loaded in Cryotop device and directly plunged into liquid nitrogen within 30 seconds.For warm interior biological temperature, the content of each vitrification device was transferred from liquid nitrogen into 200 microliter drops of decreasing trehalose solution. Oocytes were transferred set was, dragging the oocytes with a thin, glass pipette, to promote the removal of intracelular cryoprotectants. Finally, the oocytes were pre-breeded in a base medium in incubator in 5%carbon dioxide.Immediately after warming, and for each time point of post-warming culture, oocytes were morphologically evaluated using an inverted microscope with 100x magnification. The cryo tolerance of oocytes from juvenile donors is lower compared to altered ones with lower membrane integrity after warming. The use of trehalose in maturation medium induced in juvenile oocytes higher membrane integrity, increasing the survival rates after vitrification.However cleavage, fertilization, and developmental rates of juvenile oocytes were not increased by trehalose supplementation. The use of media with calcium concentration equal to 2.2 milligrams deciliter for the vitrification of juvenile oocytes, should higher fertilization rates compared to oocytes vitrified with calcium concentration, but no differences were found enabling production. By comparing post-warming culture on different durations, we showed that after four hours of culture oocyte collected from older twos are able to recover the energetic bonds and microtubular setup, and to restore the developmental competence with higher cleavage and blastocytes waste.Mitochondrial activity was higher in vitrified warm juvenile oocytes after four hours of post-warming culture, compared to other time points. Mitochondrial distribution parting also changed during six hours of post warming culture. Moreover, reactive oxygen species intracellular levels were significantly lower in juvenile oocytes at two hours of post-warming culture compared to 0, 4, and 6 hours.However, and in contrast with what found in other oocytes, the rates of spontaneous parthenogentic activation increased during post-warming culture in juvenile oocytes. One of the main advantages of the proposed method is it includes all the steps from oocyte collection to in-vitro maturation, vitrification, and warming. Moreover it includes a post-warming culture period to allow oocyte recovery from the damages incurred during the vitrification procedure.Choosing the optimum time for fertilization is challenging, and it may impact on the outcome of the vitrification program. It should also be considered that, unlike slow freezing, vitrification is a manual technique, and is thus operator dependent. For this reason, a major challenge is the need for skilled technicians and researchers who take into consideration the operator effect in the evaluation of the outcome of the vitrification program.Further studies from our lab will allow in attempting to standardize oocyte vitrification procedure and selection, and in better tailoring media composition to the needs of the juvenile oocyte. A disregard about the use of culture and character and antioxidant we offer promising opportunities.

vitrification-vitro-matured-oocytes-collected-from-adult-prepubertal

Research

JoVE Journal

Developmental Biology

4.9K visualizações.  University of Sassari. A manutenção da viabilidade potencial oócida após o armazenamento a longo prazo representa uma ferramenta de grande oportunidade, pois melhoraria a reprodução animal doméstica por meio de programas de seleção genética, contribuiria para preservar a biodiversidade através da conservação de espécies de animais selvagens e aumentaria a pesquisa em biotecnologia. A vitrificação de oócitos juvenis foi muito menor no intervalo de geração em programas de reprodução. A vitrifaç?...