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/L streptomycin (PBS).</li>
	<li>Prepare the medium for oocyte collection and maturation by diluting 9.5 g of Tissue Culture Medium (TCM) 199 in powder with 1 L of Milli-Q water supplemented with penicillin (0.1%) and streptomycin (0.1%).
	<ol>
		<li>After dilution, filter 100 mL of medium and store it at 4 &#176;C as Stock Maturation Medium (SMM) to be used for one week.</li>
		<li>Prepare the collection medium (CM) by supplementing the remaining 900 mL with 25 mM HEPES, 0.36 g/L bicarbonate and 0.1% (w/v) polyvinyl alcohol (PVA) (pH 7.3, osmolality 290 mOsm/kg).</li>
	</ol>
	</li>
	<li>Prepare the maturation medium with SMM supplemented with 0.021 g/L bicarbonate, 10% heat-treated estrus sheep serum, 1 IU/mL FSH, 1 IU/mL LH, 100 &#181;L of cysteamine and 8 mg/mL of pyruvate. 
	NOTE: The maturation medium in a volume of 10 mL must be incubated at standard conditions (in a maximum humidified atmosphere at 39 &#176;C in 5% CO2 in air) for at least 4 h before use.</li>
	<li>Prepare the base medium (BM) for manipulation of oocyte after in vitro maturation, consisting in PBS without Ca++ and Mg++, supplemented with 20% fetal calf serum (FCS).</li>
</ol>
2. Oocyte collection and maturation
<ol>
	<li>Recover the oocytes from juvenile (30-40 days of age, body weight 6-10 kg) and adult ovaries.</li>
	<li>Transport the collected ovaries from the commercial slaughterhouse to the laboratory within 1-2 h in PBS at 27 &#176;C.</li>
	<li>After washing in PBS fresh medium, slice the ovaries in CM using a micro-blade to release the follicle content.</li>
	<li>Under a stereomicroscope examination with 60x magnification, select cumulus-oocyte complexes (COCs) for in vitro maturation by choosing those with 4-10 layers of granulosa cells, oocyte with a uniform cytoplasm, homogeneous distribution of lipid droplets in the cytoplasm and with the outer diameter of about 90 &#181;m (mean).</li>
	<li>Wash the selected COCs three times in CM and finally transfer them in maturation medium. 
	NOTE: For juvenile oocytes, to improve survival after vitrification, supplement the maturation medium with 100 &#181;M trehalose.</li>
	<li>For in vitro maturation, transfer 30-35 COCs in 600 &#181;L of maturation medium in four-well Petri dishes, covered with 300 &#181;L of mineral oil and incubate them for 22 (adult oocytes)/24 (juvenile oocytes) h in 5% CO2 in air at 39 &#176;C.</li>
	<li>After in vitro maturation, denude COCs of cumulus cells by gently pipetting. Following the examination under a stereomicroscope with 60x magnification, select only those showing the extrusion on the first polar body, and thus at metaphase II (MII) stage, for vitrification.</li>
</ol>
3. Semen collection, freezing and thawing procedures
<ol>
	<li>Prepare the base medium for semen cryopreservation consisting in ram extender (200 mM Tris; 70 mM citric acid; 55 mM fructose; pH 7.2, osmolality 300 mOsm/kg) supplemented with egg yolk 20% (v/v).</li>
	<li>Collect the semen only during sheep breeding season (October-November).</li>
	<li>Obtain ejaculates by artificial vagina from adult rams (aged 2-5 years), maintained in an outdoor environment and fed a live-weight maintenance ration. Keep rams isolated in separate pens, but with visual contact between each other.</li>
	<li>Repeat semen collection one a week during the entire breeding season to obtain at least 8 ejaculates from each male.</li>
	<li>Transport the semen samples to the laboratory at environmental temperature within 5 min after collection and immediately process. Pool the ejaculates of two-three rams and evaluate sperm concentration spectrophotometry.</li>
	<li>After pooling, dilute the ejaculates up to 400 x 106 spermatozoa/mL with base medium for semen cryopreservation supplemented with 4% glycerol. Then cool the diluted semen to 4 &#176;C over a period of 2 h and equilibrate it for 20 min before freezing.</li>
	<li>Freeze the semen samples in pellet form (0.25 mL) on dry ice and then plunge them into liquid nitrogen.</li>
	<li>For thawing, put the pellet in a sterilized glass falcon and plunge it in a water bath for 20 s at 39 &#176;C.</li>
</ol>
4. In vitro fertilization and embryo culture
<ol>
	<li>Prepare stocks for constitution of the synthetical oviductal fluid (SOF).
	<ol>
		<li>Prepare Stock A: 99.4 mL of MilliQ-water; 6.29 g of NaCl; 0.534 g of KCl; 0.161 g of KH2PO4; 0.182 g of MgSO4 &#183;7H2O; and 0.6 mL of Sodium Lactate. Keep at 4 &#176;C for up to 3 months.</li>
		<li>Prepare Stock B: 10 mL of MilliQ-water; 0.210 g of NaHCO3; and 2-3 g of Phenol Red. Keep at 4 &#176;C for 1 month.</li>
		<li>Prepare Stock C: 10 mL of MilliQ-water; and 0.051 g of sodium pyruvate. Keep at 4 &#176;C for 1 month.</li>
		<li>Prepare Stock D: 10 mL of MilliQ-water; and 0.262 g of CaCl2 2H2O. Keep at 4 &#176;C for 1 month.</li>
		<li>Prepare 10 mL of SOF consisting of 7.630 mL of MilliQ water, 1 mL of Stock A, 1 mL of Stock B, 0.07 mL of Stock C and 0.7 mL of stock D.</li>
		<li>Prepare in vitro fertilization (IVF) medium: SOF supplemented with 2% heat-treated estrous sheep serum, 10 &#181;g/mL heparin and 1 &#181;g/mL hypoutarine (osmolality 280-290 mOsm/kg). 
		NOTE: The IVF medium in a volume of 10 mL must be incubated at standard conditions (in a maximum humidified atmosphere at 39 &#176;C in 5% CO2, 5% O2 and 90% N2) at least 4 h before use.</li>
	</ol>
	</li>
	<li>Transfer frozen/thawed semen aliquots in a sterilized glass conical tube below 1.5 mL of warmed IVF medium and incubate them for 15 min at 39 &#176;C in a humidified atmosphere at 5% CO2 in air.</li>
	<li>After incubation, the motile spermatozoa swim towards the apical portion of the liquid column. Collect the top layer and observe for sperm motility evaluation. 
	NOTE: Sperm motility parameters should be assessed using a computer-assisted sperm analysis (CASA) system with the following settings: 25 frames acquired to avoid sperm track overlapping, minimum contrast 10, minimum velocity of average path 30 &#181;m/s, and progressive motility &#62; 80% straightness. This system has a specific setup for ram sperm evaluation. For each sample, 5 &#181;L subsample of sperm suspension are loaded into a pre-warmed analysis chamber with a depth of 10 &#181;m. Sperm motility is assessed at 37 &#176;C at 40x using a phase contrast microscope and a minimum of 500 sperm per subsample should be analyzed in at least four different microscopic fields. The percentage of total motile and progressive motile sperm were evaluated. For the IVF, the percentage of progressive motile spermatozoa should be &#8805; 30%.</li>
	<li>Dilute swim-up derived motile spermatozoa at 1 x 106 spermatozoa/mL final concentration and co-incubate them with MII oocytes in 300 &#181;L of IVF medium covered with mineral oil in four-well Petri dishes.</li>
	<li>After 22 h transfer the presumptive zygotes in four-well Petri dishes containing SOF supplemented with 0.4% bovine serum albumin and essential and non-essential amino acids at oviductal concentration as reported by16 under mineral oil and culture them under standard conditions up to the blastocyst stage.</li>
	<li>At 22-, 26- and 32- h post-insemination, record the number of cleaved oocytes, showing two distinct blastomeres, by the examination under a stereomicroscope with 60x magnification.</li>
	<li>Observe the embryos daily starting from the fifth to the ninth day of culture and newly formed blastocysts should be recorded by the examination under a stereomicroscope with 60x magnification.</li>
</ol>
5. Oocyte vitrification and warming
NOTE: Perform vitrification following the method of minimum essential volume (MEV) using device cryotops17.
<ol>
	<li>Equilibrate a group of five oocytes at 38.5 &#176;C for 2 min in BM. The use of BM guarantees a low calcium concentration ([Ca2++] 2.2 mg/dL)10.</li>
	<li>Dehydrate the oocytes with a 3 min exposure to equilibration solution containing 7.5% (v/v) dimethyl sulfoxide (DMSO) and 7.5% (v/v) ethylene glycol (EG) in BM.</li>
	<li>Transfer the oocytes to the vitrification solution containing 16.5% (v/v) DMSO, 16.5% (v/v) EG and 0.5 M trehalose in BM before loading them in a cryotop device and directly plunging them into liquid nitrogen within 30 s.</li>
	<li>To warm to a biological temperature, transfer the content of each vitrification device from liquid nitrogen into 200 &#181;L drops of 1.25 M trehalose in BM for 1 min at 38.5 &#176;C, and gently stir to facilitate the mixing.</li>
	<li>To promote removal of intracellular cryoprotectants, transfer oocytes stepwise into 200 &#181;L drops of decreasing trehalose solutions (0.5 M, 0.25 M, 0.125 M trehalose in BM) for 30 s at 38.5 &#176;C before being equilibrated for 10 min at 38.5 &#176;C in BM.</li>
</ol>
6. Assessment of oocyte quality post-warming
<ol>
	<li>After warming, incubate the oocytes for 6 h in PBS without Ca++ and Mg++ plus 20% FCS (BM) in 5% CO2 in air at 38.5 &#176;C. 
	NOTE: The oocyte ability to restore biological and structural features after vitrification is in relation to the species and classes of used oocytes.</li>
	<li>Since the oocyte ability to recover cryopreservation damages is time-dependant, assess oocyte quality at different time points of in vitro culture (0 h, 2 h, 4 h, 6 h) after warming, to define the optimal time window for oocyte fertilization. 
	&#8203;NOTE: In adult sheep oocyte, the optimal time is 4 h post-warming; for prepubertal oocyte, the optimal time is 2 h post-warming.</li>
</ol>
7. Oocyte survival assessment
<ol>
	<li>Immediately after warming and for each time point of post-warming culture, morphologically evaluate oocytes using an inverted microscope with 100x magnification. 
	NOTE: Oocytes with structural alterations, such as faint cytoplasm, damage zona pellucida and/or membrane should be classified as degenerated.</li>
	<li>Validate the membrane integrity evaluation using a double differential fluorescent staining.</li>
	<li>Incubate the oocytes in 2 mL of BM containing propidium iodide (PI; 10 &#181;g/mL) and Hoechst 33342 (10 &#181;g/mL) for 5 min in 5% CO2 in air at 38.5 &#176;C.</li>
	<li>After washing three times in fresh BM, observe the oocytes under a fluorescent microscope using an excitation filter of 350 nm and emission of 460 nm for Hoechst 33342 and an excitation filter of 535 nm and emission of 617 nm for PI. 
	&#8203;NOTE: Oocytes with an intact membrane can be recognized by the blue fluorescence of colored DNA with Hoechst 33342. Oocyte with damaged membranes show a red fluorescence due to DNA staining with PI.</li>
</ol>
8. Evaluation of mitochondrial activity and ROS intracellular levels by confocal laser scanning microscopy
<ol>
	<li>Prepare the MitoTracker Red CM-H2XRos (MT-Red) probe.
	<ol>
		<li>Dilute the content of 1 vial (50 &#181;g) with 1 mL of DMSO to obtain a 1 mM solution. Keep the diluted vial in liquid nitrogen.</li>
		<li>Dilute the solution 1 mM with DMSO to obtain the 100 &#181;M stock solution and store it at -80 &#176;C in the dark.</li>
	</ol>
	</li>
	<li>Prepare 2&#8242;,7&#8242;-dichlorodihydrofluorescein diacetate (H2DCF-DA) probe.
	<ol>
		<li>Dilute the H2DCF-DA in 0.1% polyvinyl pyrrolidone (PVA)/PBS to obtain the first 100 mM solution. Keep the solution at -80 &#176;C in the dark .</li>
		<li>Dilute the first solution in 0.1% PVA/PBS to obtain the 100 &#181;M stock solution. Store it at -20 &#176;C in the dark.</li>
	</ol>
	</li>
	<li>Prepare the mounting medium (MM): for 10 mL of MM, add 5 mL of glycerol, 5 mL of PBS and 250 mg of sodium azide. Store it at -20 &#176;C until use.</li>
	<li>Incubate the oocytes for 30 min at 38.5 &#176;C in BM with 500 nM MT-Red (stock solution: 100 &#181;M in DMSO).</li>
	<li>After incubation with MT-Red probe, wash the oocytes three times in BM and incubate for 20 min in the same media containing 5 &#181;M H2DCF-DA (stock solution: 100 &#181;M in BM).</li>
	<li>After exposure to the probes, wash the oocytes in BM and fix in 2.5% glutaraldehyde/PBS for at least 15 min.</li>
	<li>After fixation, wash the oocytes three times in BM and mount on glass slides in a 4 &#181;L drop of MM with 1 mg/mL Hoechst 33342 using wax cushions to avoid compression of samples.</li>
	<li>Store slides at 4 &#176;C in the dark until evaluation.</li>
	<li>Perform the analysis of immunolabelled sections with a confocal laser scanning microscope. The microscope is equipped with Ar/He/Ne lasers, using a 40/60x oil objective. Analyze the sections by sequential excitation.</li>
	<li>For mitochondrial evaluation, observe the samples with a multiphoton laser to detect MT-Red (exposure: 579 nm; emission: 599 nm).</li>
	<li>Use an argon ions laser ray at 488 nm and the B-2 A filter (495 nm exposure and 519 nm emission) to point out the dichlorofluorescein (DCF)18.</li>
	<li>In each individual oocyte, measure MT-Red and DCF fluorescence intensities at the equatorial plane19.</li>
	<li>Maintain parameters related to fluorescence intensity at constant values during all image acquisitions (laser energy 26%, Sequential Settings 1: PMT1 gain 649-PMT2 gain 482; Sequential Setting 2: PMT1 gain 625-PMT2 gain 589; offset 0; pinhole size: 68).</li>
	<li>Perform quantitative analysis of fluorescence intensity using the Leica LAS AF Lite image analysis software package, following the procedures standardized by20.</li>
	<li>Capture the pictures once, moving on the Z axis, until reaching the equatorial plane.</li>
	<li>For each photo, transform to gray scale and turn off channel 1 (related to Hoechst blue) was turned off. Then manually draw a region of interest (ROI) on a circumscribed area, that is around the meiotic spindle. 
	NOTE: The software can automatically read the pixel average value on the channel 2 (FITC), subtracting the value of the background from it.</li>
	<li>Record the mean values of pixels and submit for statistical analysis.</li>
</ol>
9. Statistical Analyses
<ol>
	<li>Analyze the following differences: survival rates in juvenile vs adult oocytes, survival rates and developmental competence in control and trehalose-treated juvenile oocytes, survival and parthenogenetic activation rates and developmental competence of adult oocytes vitrified with different calcium concentration media, fertilization rates and embryo production in juvenile oocytes vitrified with low or high calcium concentration, active mitochondria phenotypes in juvenile vitrified oocytes during different time points of post-warming culture and parthenogenetic activation rates between adult and juvenile vitrified oocytes using the chi square test.</li>
	<li>Analyze the cleavage rate and embryo output in vitrified adult oocytes during different time points of post-warming culture, fluorescence intensity of mitochondrial activity and ROS intracellular levels in juvenile vitrified oocytes during different time points of post-warming culture by ANOVA after analysis for homogeneity of variance by Levene&#39;s test. Use a post-hoc test Tukey&#39;s test to highlight differences between and among groups.</li>
	<li>Perform statistical analysis using the statistical software program and consider a probability of P &#60; 0.05 to be the minimum level of significance. All results are expressed as mean &#177; S.E.M.</li>
</ol>

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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.
One of the main advantages of the proposed method is that it includes all the steps from oocyte collection, in vitro maturation, vitrification, and warming. Moreover, it includes a post-warming culture period to allow oocytes to recover from the damages incurred during the vitrification procedure before being fertilized. The optimum time for fertilization should be tailored according to the method of cryopreservation, initial oocyte quality, patient age, and species, being essential to consider both aspects of time recovery and oocyte aging25,26. Thus, choosing the duration of the post-warming incubation period is challenging and it may impact the outcome of oocyte vitrification programs. Based on the results obtained in terms of cleavage rates and embryo output, and under the conditions described in the presented protocol, the optimum time for fertilization of vitrified adult sheep oocytes is after 4 hours of post-warming incubation (Table 4)6. This information is crucial when designing an oocyte vitrification program.
This protocol, however, while giving acceptable results in terms of embryo output from vitrified/warmed adult oocytes, still leads to low to zero embryos if applied to juvenile oocytes. Several structural and functional limitations impair prepubertal oocyte developmental competence, such as small size, defective coupling between cumulus cells and oocytes, decrease in amino acid uptake, reduced protein synthesis and energy metabolism 27,28,29. In a previous study we reported that prepubertal oocytes show high sensitivity to the vitrification procedure30. The low developmental competence shown after vitrification and warming is probably the result of damages to cytoplasmic factors involved in the reorganization of the cytoskeleton and (or) in the activation of maturation promoting factor30. As shown in Table 1, the supplementation of the maturation medium with trehalose, a non-permeable cryoprotectant, was able to increase survival rates after vitrification and warming to values comparable to those of adult oocytes4. In the same way, the use of vitrification solution with low calcium concentrations increases fertilization rates of juvenile oocytes after vitrification and warming, as shown in Table 3. Thus, both the optimization on culture conditions during in vitro maturation and of the vitrification media composition may help in increasing the quality of the juvenile oocyte after vitrification and warming. Juvenile oocytes show some ability to recover from the damages induced by the vitrification procedure, as shown in Figure 3, 4 and 5.
However, the high rates of spontaneous parthenogenetic activation during post-warming culture still limit their developmental potential. Ethylene glycol and DMSO, which are commonly used cryoprotective agents, may artificially activate the oocyte before the actual fertilization, thereby limiting fertilization success and embryo development. They can indeed cause a transient increase in intracellular Ca2+ concentration31, thus triggering cortical granule exocytosis, pronuclei formation, and meiotic resumption32. In fact, vitrification may artificially activate the oocyte before the actual fertilization, thereby limiting fertilization success and embryo development. Calcium chelator may thus be used to further limit calcium availability during the vitrification process with the aim of limiting the rate of spontaneous activation in juvenile oocytes.
It should also be considered that, unlike slow-freezing, vitrification is an exclusively manual technique and it is thus operator dependent33,34. Thus, the availability of trained personnel is a key factor for the success of this method. First of all, the operator has to properly select the oocytes to be vitrified. After IVM, oocytes are gently denuded of cumulus cells and evaluated under a stereomicroscope to select for cryopreservation only those with a uniform cytoplasm, homogeneous distribution of lipid droplets in the cytoplasm and with the outer diameter of about 90 &#181;m. Moreover, only oocytes showing the extrusion on the first polar body, and thus at the MII stage, must be selected.
The morphological evaluation of the oocytes must be completed in a few minutes and being operator-dependant, it is very sensitive to variations in its proper implementation. To help standardize the selection procedure, the method suggests limiting the culture time for in vitro maturation to 22 h for adult oocytes. At this time point, sheep oocytes of high quality have already completed the first meiotic division22 and can be selected for cryopreservation. This way the elimination of low-quality oocytes, which are the slowest ones in the completion of the first meiotic division, should be simpler.
The operator must also strictly respect the timing set for the vitrification procedure, from the first exposure to the cryoprotectant to the immersion in liquid nitrogen. Another critical step is the loading of the oocyte in the vitrification device used. The procedure must use minimum sample volumes to increase the cooling rate and to help cells pass through the phase transition temperature rapidly, thereby decreasing cryoinjuries35. Cryotop uses a polypropylene strip attached to a holder. In this method, the oocytes in the vitrification solution (&#60;0.1 &#181;L) are rapidly loaded with a glass capillary on top of the film strip. Then, the solution must be removed, leaving behind a thin layer sufficient to cover the cells to be cryopreserved. Once again, this step must be completed as fast as possible to limit the exposure of the oocytes to the high cryoprotectant concentrations of the vitrification solution, which can cause osmotic shock and are toxic to the cells.
For these reasons, a major challenge associated with this method is the need for manual handling and skilled technician. Other authors reported that the oocyte vitrification outcome appears influenced by a &#34;learning curve&#34; effect, as the acquisition of manual skills can significantly reduce the biological damage induce by the vitrification procedure34. Researchers should thus take into consideration the &#34;operator effect&#34; in the evaluation of the outcome of the vitrification procedure.
Further studies will focus both on standardizing the oocyte selection procedure and better tailoring media composition and culture conditions to the needs of the juvenile oocytes. At this regard, both the use of calcium chelator and antioxidants may offer promising opportunities. Similarly, the optimization of the protocol will allow increasing the developmental competence of vitrified/warmed adult oocytes.

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><tbody><tr><td>2&amp;prime;,7&amp;prime;-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 (MgSO&lt;sub&gt;4&lt;/sub&gt;.7H&lt;sub&gt;2&lt;/sub&gt;O)</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-H&lt;sub&gt;2&lt;/sub&gt;XRos</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 (KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;)</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 (NaHCO&lt;sub&gt;3)&lt;/sub&gt;</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 demonstrated23 that oocytes matured for 24 h with trehalose supplementation showed higher survival rates after vitrification/warming compared to the non-treated group (Table 1: 85.7% vs 75.3% respectively; &#967;2 test P&#60;0.05). Trehalose supplementation was indeed associated with higher membrane integrity after warming (Figure 2A). Thus, the use of trehalose during the in vitro maturation of juvenile oocytes increased the rates of survival after vitrification (85.7%) to values comparable to adult ones (90.3%). However, cleavage, fertilization and developmental rates of juvenile oocytes were not increased by trehalose supplementation (Table 1).
To improve oocyte competence after vitrification we tested in adult ovine oocytes different vitrification media with calcium concentrations ranging from 9.9 to 0.4 mg/dL10. Obtained results showed that the use of media with calcium concentration equal to 2.2 mg/dL increased post-warming survival rates, improved developmental competence and reduced parthenogenetic activation of adult oocyte10 (Table 2). We thus tested the low calcium vitrification media for the vitrification of juvenile oocytes. As shown in Table 3, juvenile oocytes vitrified with low calcium concentration evidenced higher fertilization rates compared to oocytes vitrified with high calcium concentration (44.35% vs 32.29 % respectively; P&#60;0.05), but no differences were found in embryo production.
Vitrified/warmed oocytes need extra time prior to fertilization to restore damage due to cryopreservation procedures and to increase their developmental potential. A previous study has indeed demonstrated that ATP intracellular concentration, mitochondrial activity and in vitro developmental competence are reduced in vitrified/thawed oocytes, which also show high intracellular ROS concentrations6. These alterations are particularly marked immediately after warming. During the post-warming culture, both adult and juvenile oocytes are able to partially recover from the damages suffered during the vitrification procedures6, 24. By comparing post-warming culture of different durations (0, 2, 4, and 6 h), we showed that after 4 h of culture oocytes collected from adult ewes are able to recover the energetic balance6 and microtubular setup24 and to restore the developmental competence with higher cleavage (50.7 &#177; 3.9%; P&#60; 0.01 ANOVA) and blastocyst rates (14.40 &#177; 1.3%; ANOVA P&#60; 0.01) compared to other time points (0, 2 and 6 h; Table 4). Thus, 4 h of post-warming culture represents the ideal time window for fertilization of vitrified/warmed adult oocytes6.
When the same experiment was repeated with oocytes collected from juvenile donors, these results were partially confirmed. Mitochondrial activity was higher in vitrified/warmed juvenile oocytes after 4 h of post-warming culture compared to other time points (0, 2, 6; Figure 3: ANOVA P&#60;0.01). Several patterns of mitochondrial distribution are observed and classified into the following three groups (as reported by21 ): Pattern A: homogeneous FINE with small granulations spread throughout the cytoplasm; Pattern B: homogeneous GRANULAR with large granulations spread throughout the cytoplasm; Pattern C: heterogeneous CLUSTERED when particularly large granulations were present, spread all over the cytoplasm or located in specific cytoplasmic domains. The different phenotypes in the cytoplasm distribution of active mitochondrial in MII can be related to oocyte developmental competence. A FINE homogeneous distribution is an indicator of poor developmental competence while a GRANULAR and CLUSTERED distribution are related to an increased mitochondria activity and consequently higher developmental competence22. Mitochondrial distribution patterns changed during 6 h of post-warming culture. Figure 4 shows examples of juvenile oocytes having different patterns of mitochondrial distribution and their fluctuations during post-warming culture. The pattern A increase significantly during the prolonged incubation and reaches the higher value at 6 h post-warming (Figure 4Aa: &#967;2 P&#60;0.05), the pattern B did not show significant changes during post-warming culture (Figure 4Bb), the pattern C was not found in any juvenile vitrified/warmed oocyte during the prolonged incubation (Figure 4Cc: &#967;2 P&#60;0.05).
Moreover, ROS intracellular levels were significantly lower in juvenile oocytes at 2 h of post-warming culture compared to 0, 4 and 6 h (Figure 5: ANOVA P&#60;0.001). However, and in contrast to what was found in adult oocytes, the rate of spontaneous parthenogenetic activation increased during the post-warming culture in juvenile oocytes (Figure 6). For this reason, the recommended time point for fertilization in juvenile oocytes would be 2 h after the post warming culture.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62272/62272fig01.jpg" /> 
Figure 1. Survival rates of vitrified/warmed ovine oocyte collected from juvenile and adult donors. (A) Oocyte were vitrified after in vitro maturation. Survival rates were determined after vitrification and warming by fluorescent staining with propidium iodide (10 &#181;g/mL) and Hoechst 33342 (10 &#181;g/mL). N = 165 adult oocytes and 170 juvenile oocytes. Different letters indicate significant differences between adult and juvenile oocytes: &#967;2 test P&#60;0.001. (B-C) Examples of vitrified/warmed juvenile oocytes with intact (B) and damaged plasma membrane (C) at the morphological evaluation (inverted microscope with 100x magnification). Scale bar = 10 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62272/62272fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62272/62272fig02.jpg" /> 
Figure 2. Juvenile ovine oocyte survival rates and in vitro developmental competence after maturation with (TRH) and without (CTR) trehalose (100 mM in maturation medium). (A-B) Examples of juvenile oocytes vitrified after in vitro maturation in media supplemented (A) with or (B) without trehalose at the morphological evaluation (inverted microscope with 100x magnification). Scale bar = 30 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62272/62272fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62272/62272fig03.jpg" /> 
Figure 3. Quantification of active mitochondrial fluorescence intensity in vitrified/warmed juvenile oocytes at different time points (0, 2, 4, 6) during post-warming in vitro culture. IVM oocytes were used as a control (CTR N = 77). In total 163 (0 h N = 45; 2 h N = 39; 4 h N = 40; 6 h N = 39) juvenile oocytes were vitrified and warmed in three independent experiments. Different letters indicate statistically significant differences (ANOVA P = 0.0000). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62272/62272fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62272/62272fig04.jpg" /> 
Figure 4. Distribution of mitochondrial pattern in vitrified/warmed juvenile oocytes during 6 hours of post-warming in vitro culture. Representative images of Fine (A), Granular (B) and Clustered (C) mitochondrial distribution in vitrified/warmed juvenile oocytes. (D) Percentage of juvenile vitrified/warmed oocytes showing a fine mitochondrial distribution; (E) Percentage of juvenile vitrified/warmed oocytes showing a granular mitochondrial distribution; (F) Percentage of juvenile vitrified/warmed oocytes showing a clustered mitochondrial distribution. IVM juvenile oocytes were used as control (CTR; N = 77). In total 163 (0 h N = 45; 2 h N = 39; 4 h N = 40; 6 h N = 39) juvenile oocytes vitrified and warmed in three independent experiments were used. Different letters indicate statistically significant differences (Aa: &#967;2 P = 0.026; Bb: &#967;2 P = 0.097; Cc: &#967;2 P = 0.014). Scale bar = 30 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62272/62272fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/62272/62272fig05.jpg" /> 
Figure 5. Quantification of intracellular ROS fluorescence intensity in vitrified juvenile oocytes during of 6 hours post-warming in vitro culture. (A-B) Representative images of ROS fluorescence intensity in in vitro matured (A) and vitrified (B) juvenile oocytes. (C) Intracellular levels of ROS as determined by quantification of fluorescence intensity in vitrified juvenile oocytes at different time points (0 h N=45; 2 h N=39; 4 h N=40; 6 h N=39) during post-warming in vitro culture. In vitro matured juvenile oocytes were used as a control (CTR N = 77). Different letters indicate statistically significant differences (ANOVA P = 0.0000). Scale bar = 50 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62272/62272fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/62272/62272fig06.jpg" /> 
Figure 6. Parthenogenetic activation in vitrified juvenile and adult oocytes during 6 hours of post-warming in vitro culture. (A-B) Representative images of oocyte parthenogenetic activation: (A) oocyte in metaphase II-telophase II transition and (B)&#160;pronucleus formation. (C) Percentages of parthenogenetic activated adult and juvenile oocytes at different time points (0, 2, 4, 6 h) during post-warming in vitro culture. Asterisks indicate statistical differences between juvenile and adult oocytes at each time point of incubation (ANOVA P = 0.000). This figure has been modified from Serra et al.24 Scale bar = 50 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62272/62272fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>Oocytes (n)</td>
			<td>Survival rate (%)</td>
			<td>IVF (n)</td>
			<td>Fertilizeda (%)</td>
			<td>Cleavedb (%)</td>
			<td>Blastocystsc (%)</td>
		</tr>
		<tr>
			<td>CTR</td>
			<td>73</td>
			<td>75.3*</td>
			<td>452</td>
			<td>91.1</td>
			<td>96.1</td>
			<td>14.3</td>
		</tr>
		<tr>
			<td>TRH</td>
			<td>77</td>
			<td>85.7</td>
			<td>470</td>
			<td>92.5</td>
			<td>95.4</td>
			<td>13</td>
		</tr>
		<tr>
			<td colspan="7">* Chi square test p&#60;0.5</td>
		</tr>
		<tr>
			<td colspan="7">a Percentages are calculated on IVF oocytes</td>
		</tr>
		<tr>
			<td colspan="7">b Percentages are calculated on fertilized oocytes</td>
		</tr>
		<tr>
			<td colspan="7">c Percentages are calculated on cleaved oocytes</td>
		</tr>
	</tbody>
</table>
Table 1.&#160;Juvenile ovine oocytes survival rates and in vitro developmental competence after maturation with and without trehalose and vitrification. TRH = juvenile oocytes matured with trehalose supplementation (100 mM in maturation medium). CTR = control juvenile oocytes matured without trehalose supplementation. Survival rates were determined after fluorescent staining with propidium iodide (10 &#181;g/mL) and Hoechst 33342 (10 &#181;g/mL) of vitrified/warmed oocytes. Oocyte developmental competence was determined after incorporation in an in vitro production system. a Percentages are calculated on IVF oocytes. b Percentages are calculated on fertilized oocytes. c Percentages are calculated on cleaved oocytes. * &#967;2 test P&#60;0.5. This table has been modified from Berlinguer et al.23
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Groups</td>
			<td>[Ca++] mg/dL</td>
			<td>N oocyte</td>
			<td>Spontaneous parthenogenetic activation (%)</td>
			<td>Vitrified oocytes</td>
			<td>Survived and IVF oocytes (%)</td>
			<td>Cleavage (%)</td>
			<td>Blastocysts output (%)</td>
		</tr>
		<tr>
			<td>TCM/FCS</td>
			<td>9.9</td>
			<td>80</td>
			<td>33 (41.2)a</td>
			<td>150</td>
			<td>124 (82.7)a</td>
			<td>40 (32.5)a</td>
			<td>2 (1.6)a</td>
		</tr>
		<tr>
			<td>PBS/FCS</td>
			<td>4.4</td>
			<td>82</td>
			<td>29 (35.3)ac</td>
			<td>115</td>
			<td>88 (76.5)a</td>
			<td>33 (37.5)ae</td>
			<td>1 (1.1)a</td>
		</tr>
		<tr>
			<td>PBSCaMg free/FCS</td>
			<td>2.2</td>
			<td>86</td>
			<td>11 (12.7)b</td>
			<td>126</td>
			<td>115 (91.3)b</td>
			<td>74 (64.3)b</td>
			<td>12 (10.4)b</td>
		</tr>
		<tr>
			<td>PBS/BSA</td>
			<td>3.2</td>
			<td>83</td>
			<td>21 (25.3)c</td>
			<td>110</td>
			<td>90 (81.8)a</td>
			<td>18 (20)c</td>
			<td>0 (0)a</td>
		</tr>
		<tr>
			<td>PBSCaMg free/BSA</td>
			<td>0.4</td>
			<td>87</td>
			<td>10 (11.5)b</td>
			<td>149</td>
			<td>123 (82.5)a</td>
			<td>57 (46.3)de</td>
			<td>3 (2.4)b</td>
		</tr>
	</tbody>
</table>
Table 2. Developmental competence of in vitro matured adult oocytes vitrified in vitrification media (16.5% ethylene glycol + 16.5% dimethyl sulfoxide) containing different calcium concentrations. Survival and fertilization rates are calculated on vitrified oocytes; total cleavage and blastocyst rates are calculated on survived oocytes. Values with different subscript within the same column are significantly different: &#967;2 test P&#60;0.05. This table has been modified from Succu et al.10
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>[Ca 2++] in vitrification media</td>
			<td>No. Oocytes</td>
			<td>Post-vitrification survival rate (%)</td>
			<td>Fertilization rate (%)</td>
			<td>Cleavage (%)</td>
			<td>Blastocyst output (%)</td>
		</tr>
		<tr>
			<td>High [9.9 mg/dL]</td>
			<td>190</td>
			<td>161 (84.73)</td>
			<td>52/161 (32.29)a</td>
			<td>43/161 (26.7)</td>
			<td>0</td>
		</tr>
		<tr>
			<td>Low [2.2 mg/dL]</td>
			<td>150</td>
			<td>124 (82.66)</td>
			<td>55/124 (44.35)b</td>
			<td>41/124 (33.1)</td>
			<td>0</td>
		</tr>
	</tbody>
</table>
Table 3. Fertilization and developmental rates after in vitro fertilization and culture of vitrified/warmed juvenile oocyte using high ([Ca 2++] = 9.9 mg/dL) and low ([Ca 2++] = 2.2 mg/dL) calcium concentration in vitrification media. Different letters indicate statistical difference (a &#8800; b P&#60;0.05 &#967;2 test).
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Hours of post-warming incubation</td>
			<td>Cleavage rate (n)</td>
			<td>Embryo output (n)</td>
		</tr>
		<tr>
			<td>0</td>
			<td>19.2 &#177; 3%a (82)</td>
			<td>0%a (17)</td>
		</tr>
		<tr>
			<td>2</td>
			<td>41.8 &#177; 3%b (100)</td>
			<td>6.5 &#177; 1.3%b (42)</td>
		</tr>
		<tr>
			<td>4</td>
			<td>50.7 &#177; 3%b (92)</td>
			<td>14.4 &#177; 1.3%c (48)</td>
		</tr>
		<tr>
			<td>6</td>
			<td>26 &#177; 3%a (92)</td>
			<td>0%a (23)</td>
		</tr>
	</tbody>
</table>
Table 4. Cleavage rate and embryo output in vitrified/warmed adult oocytes fertilized at different time points of post-warming culture. Different letters indicate statistical difference within the same column: ANOVA P&#60;0.01.

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 ...

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

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

5.1K Views.  University of Sassari. Het behoud van de potentiële levensvatbaarheid van oöcyt na langdurige opslag is een instrument van grote kansen, omdat het de fokkerij van huisdieren zou verbeteren door genetische selectieprogramma's, zou bijdragen aan het behoud van de biodiversiteit door het behoud van wilde diersoorten en het onderzoek in de biotechnologie zou vergroten. Juveniele oöcyt vitrificatie was een stuk korter in het generatie-interval in fokprogramma's. Vitrifactie door ultrarapide koeling en opwarming wordt...

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

Culture and Co-Culture of Mouse Ovaries and Ovarian Follicles

The mammalian ovary is composed of ovarian follicles, each follicle consisting of a single oocyte surrounded by somatic granulosa cells, enclosed together within a basement membrane. A finite pool of follicles is laid down during embryonic development, when oocytes in meiotic arrest form a close association with flattened granulosa cells, forming primordial follicles. By or shortly after birth, mammalian ovaries contain their lifetime&#8217;s supply of primordial follicles, from which point onwards there is a steady release of follicles into the growing follicular pool.
The ovary is particularly amenable to development in vitro, with follicles growing in a highly physiological manner in culture. This work describes the culture of whole neonatal ovaries containing primordial follicles, and the culture of individual ovarian follicles, a method which can support the development of follicles from an immature through to the preovulatory stage, after which their oocytes are able to undergo fertilization in vitro. The work outlined here uses culture systems to determine how the ovary is affected by exposure to external compounds. We also describe a co-culture system, which allows investigation of the interactions that occur between growing follicles and the non-growing pool of primordial follicles.

This protocol describes the primary culture/co-culture of mouse ovarian tissue, using ovaries from neonatal mice and individual ovarian follicles from prepubertal mice. The culture techniques support development in a highly physiological manner, allowing investigation of the effect of extrinsic agents on the ovary, and of interactions between ovarian follicles.

The mammalian ovary is composed of ovarian follicles, each follicle consisting of a single oocyte surrounded by somatic granulosa cells, enclosed together ...

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 ...

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.

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 ...

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 ...

Ovarian Tissue Oocyte-In Vitro Maturation for Fertility Preservation

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

Ovarian Tissue Oocyte-In Vitro Maturation for Fertility Preservation

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

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

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

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

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

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

Medicine

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

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

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

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

Fertility Preservation Through Oocyte Vitrification: Clinical and Laboratory Perspectives

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

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

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

Proteolytically Degraded Alginate Hydrogels and Hydrophobic Microbioreactors for Porcine Oocyte Encapsulation

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

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

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

Biology

Follicular Viability Assessment in Vitrified Human Ovarian Cortex Tissues

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

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

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

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

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

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

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