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

这项工作得到了EVSSAR（欧洲小动物繁殖兽医学会）2016年赠款和米兰大学，钢琴迪索斯特格诺 alla Ricerca 2019（Linea 2 Azione A）的部分支持。我们还要感谢MariaGiorgiaMorselli博士对特此描述的实验和图像采集的贡献。

特此描述的程序没有经过道德批准，因为猫卵巢是在兽医诊所收集的，作为从主人要求的常规卵巢切除术或卵巢切除术的副产品收集的。
1. 卵母细胞集合
<ol><li>在开始实验之前，为卵巢和卵母细胞的收集准备解决方案。用磷酸盐缓冲盐水 （PBS） 制备卵巢收集溶液，使用抗生素和抗霉药的混合物（100 IU/mL 青霉素 G 钠，0.1 mg/mL 硫酸链霉素，0.25 μg/mL 的氨基霉素 B）。使用 PBS ?...

<ol>
	<li>Bankowski, B., Lyerly, A., Faden, R., Wallach, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+social+implications+of+embryo+cryopreservation.">The social implications of embryo cryopreservation.</a> Fertility and Sterility. 84 (4), 823-832 (2005).</li><li>Saragusty, J., Arav, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+progress+in+oocyte+and+embryo+cryopreservation+by+slow+freezing+and+vitrification.">Current progress in oocyte and embryo cryopreservation by slow freezing and vitrification.</a> Reproduction. 141 (1), 1-19 (2011).</li><li>Luvoni, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gamete+cryopreservation+in+the+domestic+cat.">Gamete cryopreservation in the domestic cat.</a> Theriogenology. 66 (1), 101-111 (2006).</li><li>Thongphakdee, A., Tipkantha, W., Punkong, C., Chatdarong, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+and+controlling+ovarian+activity+in+wild+felids.">Monitoring and controlling ovarian activity in wild felids.</a> Theriogenology. 109, 14-21 (2018).</li><li>Parks, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypothermia+and+mammalian+gametes.">Hypothermia and mammalian gametes.</a> Reproductive Tissue Banking. , 229-261 (1997).</li><li>Carabatsos, M. J., Sellitto, C., Goodenough, D. A., Albertini, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oocyte-granulosa+cell+heterologous+gap+junctions+are+required+for+the+coordination+of+nuclear+and+cytoplasmic+meiotic+competence.">Oocyte-granulosa cell heterologous gap junctions are required for the coordination of nuclear and cytoplasmic meiotic competence.</a> Developmental Biology. 226 (2), 167-179 (2000).</li><li>Murakami, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blastocysts+derived+from+in+vitro-fertilized+cat+oocytes+after+vitrification+and+dilution+with+sucrose.">Blastocysts derived from in vitro-fertilized cat oocytes after vitrification and dilution with sucrose.</a> Cryobiology. 48 (3), 341-348 (2004).</li><li>Merlo, B., Iacono, E., Regazzini, M., Zambelli, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cat+blastocysts+produced+in+vitro+from+oocytes+vitrified+using+the+cryoloop+technique+and+cryopreserved+electroejaculated+semen.">Cat blastocysts produced in vitro from oocytes vitrified using the cryoloop technique and cryopreserved electroejaculated semen.</a> Theriogenology. 70 (1), 126-130 (2008).</li><li>Luciano, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+different+cryopreservation+protocols+on+cytoskeleton+and+gap+junction+mediated+communication+integrity+in+feline+germinal+vesicle+stage+oocytes.">Effect of different cryopreservation protocols on cytoskeleton and gap junction mediated communication integrity in feline germinal vesicle stage oocytes.</a> Cryobiology. 59 (1), 90-95 (2009).</li><li>Comizzoli, P., Wildt, D. E., Pukazhenthi, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+compaction+of+germinal+vesicle+chromatin+is+beneficial+to+survival+of+vitrified+cat+oocytes.">In vitro compaction of germinal vesicle chromatin is beneficial to survival of vitrified cat oocytes.</a> Reproduction in Domestic Animals. 44, 269-274 (2009).</li><li>Alves, A. E., Kozel, A. C., Luvoni, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vitrification+with+DAP+213+and+Cryotop+of+ex+situ+and+in+situ+feline+cumulus-oocyte+complexes.">Vitrification with DAP 213 and Cryotop of ex situ and in situ feline cumulus-oocyte complexes.</a> Reproduction in Domestic Animals. 47 (6), 1003-1008 (2012).</li><li>Kuwayama, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+efficient+vitrification+for+cryopreservation+of+human+oocytes+and+embryos:+The+Cryotop+method.">Highly efficient vitrification for cryopreservation of human oocytes and embryos: The Cryotop method.</a> Theriogenology. 67 (1), 73 (2007).</li><li>Pope, C. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+survival+of+domestic+cat+oocytes+after+vitrification,+intracytoplasmic+sperm+injection+and+embryo+transfer.">In vivo survival of domestic cat oocytes after vitrification, intracytoplasmic sperm injection and embryo transfer.</a> Theriogenology. 77 (3), 531-538 (2012).</li><li>Galiguis, J., G&#243;mez, M. C., Leibo, S. P., Pope, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Birth+of+a+domestic+cat+kitten+produced+by+vitrification+of+lipid+polarized+in+vitro+matured+oocytes.">Birth of a domestic cat kitten produced by vitrification of lipid polarized in vitro matured oocytes.</a> Cryobiology. 68 (3), 459-466 (2014).</li><li>Fernandez-Gonzalez, L., Jewgenow, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryopreservation+of+feline+oocytes+by+vitrification+using+commercial+kits+and+slush+nitrogen+technique.">Cryopreservation of feline oocytes by vitrification using commercial kits and slush nitrogen technique.</a> Reproduction in Domestic Animals. 52, 230-234 (2017).</li><li>Colombo, M., Morselli, M. G., Tavares, M. R., Apparicio, M., Luvoni, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+competence+of+domestic+cat+vitrified+oocytes+in+3D+enriched+culture+conditions.">Developmental competence of domestic cat vitrified oocytes in 3D enriched culture conditions.</a> Animals. 9 (6), 329 (2019).</li><li>Colombo, M., Morselli, M. G., Apparicio, M., Luvoni, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Granulosa+cells+in+three-dimensional+culture:+A+follicle-like+structure+for+domestic+cat+vitrified+oocytes.">Granulosa cells in three-dimensional culture: A follicle-like structure for domestic cat vitrified oocytes.</a> Reproduction in Domestic Animals. , (2020).</li><li>Wood, T. C., Wildt, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+the+quality+of+the+cumulus-oocyte+complex+in+the+domestic+cat+on+the+ability+of+oocytes+to+mature,+fertilize+and+develop+into+blastocysts+in+vitro.">Effect of the quality of the cumulus-oocyte complex in the domestic cat on the ability of oocytes to mature, fertilize and develop into blastocysts in vitro.</a> Journal of Reproduction and Fertility. 110 (2), 355-360 (1997).</li><li>Cobo, A., Kuwayama, M., P&#233;rez, S., Ruiz, A., Pellicer, A., Remoh&#237;, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+concomitant+outcome+achieved+with+fresh+and+cryopreserved+donor+oocytes+vitrified+by+the+Cryotop+method.">Comparison of concomitant outcome achieved with fresh and cryopreserved donor oocytes vitrified by the Cryotop method.</a> Fertility and Sterility. 89 (6), 1657-1664 (2008).</li><li>Mullen, S. F., Critser, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+science+of+cryobiology.">The science of cryobiology.</a> Oncofertility - Fertility preservation for Cancer Survivors. , 83-109 (2007).</li><li>Fahy, G. M., Wowk, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+of+cryopreservation+by+vitrification.">Principles of cryopreservation by vitrification.</a> Methods in Molecular Biology. 1257, 21-82 (2015).</li><li>Apparicio, M., Ruggeri, E., Luvoni, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vitrification+of+immature+feline+oocytes+with+a+commercial+kit+for+bovine+embryo+vitrification.">Vitrification of immature feline oocytes with a commercial kit for bovine embryo vitrification.</a> Reproduction in Domestic Animals. 48 (2), 240-244 (2013).</li><li>Brambillasca, F., Guglielmo, M. C., Coticchio, G., Mignini Renzini, M., Dal Canto, M., Fadini, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+current+challenges+to+efficient+immature+oocyte+cryopreservation.">The current challenges to efficient immature oocyte cryopreservation.</a> Journal of Assisted Reproduction and Genetics. 30 (12), 1531-1539 (2013).</li><li>Moussa, M., Shu, J., Zhang, X., Zeng, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryopreservation+of+mammalian+oocytes+and+embryos:+current+problems+and+future+perspectives.">Cryopreservation of mammalian oocytes and embryos: current problems and future perspectives.</a> Science China Life Sciences. 57 (9), 903-914 (2014).</li><li>Luvoni, G., Pellizzari, P., Battocchio, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+slow+and+ultrarapid+freezing+on+morphology+and+resumption+of+meiosis+in+immature+cat+oocytes.">Effects of slow and ultrarapid freezing on morphology and resumption of meiosis in immature cat oocytes.</a> Journal of Reproduction and Fertility. Supplement. 51, 93-98 (1997).</li><li>Calvo, J. J., Robert, D., Viqueira, M., Lombide, P., Calvo, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vitrified+and+in+vitro+matured+oocytes+viability+from+adult+housecat+(Felis+catus)+in+reproductive+season.">Vitrified and in vitro matured oocytes viability from adult housecat (Felis catus) in reproductive season.</a> International Journal of Morphology. 33 (4), (2015).</li><li>Nowak, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+viability+of+Serval+(Leptailurus+serval)+and+Pallas+Cat+(Felis+manul)+oocytes+after+cryopreservation+using+the+Rapid-I+Method.">The viability of Serval (Leptailurus serval) and Pallas Cat (Felis manul) oocytes after cryopreservation using the Rapid-I Method.</a> Cryo Letters. 40 (4), 226-230 (2019).</li></ol>

卵母细胞冷冻保存是一种关键的种质保护技术，特别是在许多物种濒临灭绝的分类中，如费利达家族。在这份手稿中，提出了一个简单和现场友好的协议，用于对未成熟的猫卵母细胞进行玻璃化。实验室制造的介质、最小体积的玻璃化支持和训练有素的人员是此方法成功的关键因素，它允许持续和反复地获得可行的卵母细胞，特此报告的代表性结果所示。
从媒体准备开始，?...

冷冻保存已成为辅助生殖技术（ART）的关键步骤。在人类中，它允许保留生育能力或因医疗或个人原因推迟为人父母。在动物中，有必要克服距离和时间在计划交配，特别是在农场动物和宠物，或保存遗传物质的宝贵主题在保护计划，特别是在野生濒危物种。游戏冷冻保存是最佳选择，当被培育的个体尚未被选中或为了避免与胚胎冷冻相关的伦理问题，特别是在人类医学1。Spermatozoa 相对容易保存，解冻后给出令人满意的结果，但由于卵母细胞的结构特征，其储存可能更为复杂。事实上，低表面/体积比，以及围绕乌普拉斯玛的佐纳佩普西达的存在，限制了低温保护物和水在细胞2的移动。此外，在家畜，包括幸福动物，他们的特点是脂质丰富的细胞质，这被认为是使他们更敏感的冷冻保存3。
大多数猫都受到威胁，由于常规的奥瓦里切除术提供性腺，家猫被用作开发种质保存协议的模型。在野生动物中，性腺可以在选择性手术后或（更频繁地）验尸后获得，并且可以检索到不成熟的（胚芽囊泡）游戏物。激素刺激旨在获得成熟（元相II）卵母细胞是不太常见的人类ART，因为伦理问题和物种和个人特异性反应治疗4。
因此，低温保存策略的发展侧重于不成熟的配子，这些配子通常可以在罕见个体意外或突然死亡后被检索。从生物学的角度来看，不成熟或成熟的配子的低温保存存在一些差异，每种游戏体都有其优点。首先，DNA在未成熟的卵母细胞中受到更多的保护，其胚芽囊囊含有被核膜包围的染色体，而元相II卵母细胞的卵母体主轴可能更容易受到低温损伤5。其次，冷诱发的细胞骨架损伤可能会影响主轴旋转、极体挤压、亲核迁移和细胞因子，根据卵母细胞发育阶段，它们可能产生不同的影响，影响美因病进展或受精后事件。最后，也许最重要的是，虽然成熟的卵母细胞已准备好受精，但不成熟的卵母细胞依靠周围积细胞的支持来度过核和细胞质成熟6，这就是为什么整个积细胞-卵母细胞复合物（COCs）是冷冻保存的原因。然而，累积细胞的丧失和/或游戏细胞与周围体细胞之间功能连接的丧失可能是未成熟COCs的低温保存的最有害影响。
在低温保存技术中，玻璃化是一种在野外条件下更容易应用的技术。与缓慢（或受控速率）冷冻相比，玻璃化速度更快，不需要特定的设备，如可编程冰柜。为了满足玻璃化的三个基本原则（即高粘度，与高低温保护浓度、体积小和超快速温度降低有关），在猫中特别开发和使用了几种介质和支架，用于成熟和成熟的卵母细胞。从简单的吸管7开始，设备被开发，以达到"最小体积"的目标。Cryoloop8，开拉吸管（OPS）9，塑料排水沟（改性吸管）10和冷冻管11已经使用，直到一个更高效的装置（即冷冻站）被采用11，改善生存和梅病恢复。Cryotop（ 补充图 1） 是一种市售支持， 已成为玻璃化的选修开放系统.它专为人类卵母细胞和胚胎的玻璃化而开发，由附着在硬塑料支架上的小型薄膜条组成，在储存12期间由塑料管盖保护。由于其可用性和极小的玻璃化体积（低至0.1μL），这也导致极快的冷却和变暖率，这种玻璃化支持已越来越多地应用于几个物种，包括家猫，其中它已被用于各种媒体13，14，15，16，17。13,14,15,16,17
本手稿的目的是描述一个收集-玻璃化-加热协议，与最初为人类卵母细胞开发的，使用实验室制造的介质和商业支持，以最小体积的玻璃化，可以很容易地应用于现场条件，为不成熟的猫科动物的低温保存。

<table>
	<tbody>
		<tr>
			<td>Amphotericin B</td>
			<td>Sigma-Aldrich</td>
			<td>A2942</td>
			<td>/</td>
		</tr>
		<tr>
			<td>Automatic pipettes &#38; tips</td>
			<td>/</td>
			<td>/</td>
			<td>Needed to pipette 20, 100, 240 and 300 &#181;L</td>
		</tr>
		<tr>
			<td>Surgical scalpels</td>
			<td>/</td>
			<td>/</td>
			<td>Size 10 is usually ok</td>
		</tr>
		<tr>
			<td>Bunsen beak</td>
			<td>/</td>
			<td>/</td>
			<td>/</td>
		</tr>
		<tr>
			<td>Clamps</td>
			<td>/</td>
			<td>/</td>
			<td>Some small (Mosquito clamp) for ovary isolation, some bigger (Klemmer clamp) to work in liquid nitrogen</td>
		</tr>
		<tr>
			<td>Cryotop</td>
			<td>Kitazato (distributor: MBT - Medical Biological Technologies)</td>
			<td>01.CR</td>
			<td>Distributors and catalog number may change in different countries</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Sigma-Aldrich</td>
			<td>D2650</td>
			<td>/</td>
		</tr>
		<tr>
			<td>Ethylene glycol (EG)</td>
			<td>Sigma-Aldrich</td>
			<td>E9129</td>
			<td>/</td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Sigma-Aldrich</td>
			<td>F9665</td>
			<td>/</td>
		</tr>
		<tr>
			<td>Glass Pasteur pipettes</td>
			<td>/</td>
			<td>/</td>
			<td>Advised lenght 230 mm (100+130)</td>
		</tr>
		<tr>
			<td>Gobelets</td>
			<td>/</td>
			<td>/</td>
			<td>According to the canisters of the storage tank</td>
		</tr>
		<tr>
			<td>Heating stage</td>
			<td>/</td>
			<td>/</td>
			<td>All heating stages are ok, as long as they can keep 38&#186;C</td>
		</tr>
		<tr>
			<td>Liquid nitrogen</td>
			<td>/</td>
			<td>/</td>
			<td>/</td>
		</tr>
		<tr>
			<td>Medium 199</td>
			<td>Sigma-Aldrich</td>
			<td>M4530</td>
			<td>/</td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline (PBS)</td>
			<td>Sigma-Aldrich</td>
			<td>D8662</td>
			<td>/</td>
		</tr>
		<tr>
			<td>Penicillin G sodium</td>
			<td>Sigma-Aldrich</td>
			<td>P3032</td>
			<td>/</td>
		</tr>
		<tr>
			<td>Polyvinyl alcohol (PVA)</td>
			<td>Sigma-Aldrich</td>
			<td>P8136</td>
			<td>/</td>
		</tr>
		<tr>
			<td>Repro plate</td>
			<td>Kitazato (distributor: MBT - Medical Biological Technologies)</td>
			<td>01.K-2</td>
			<td>Distributors and catalog number may change in different countries</td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>/</td>
			<td>/</td>
			<td>As long as the operator can select the oocytes, other stereomicroscopes are ok</td>
		</tr>
		<tr>
			<td>Storage tank</td>
			<td>/</td>
			<td>/</td>
			<td>Any regularly filled tank is ok</td>
		</tr>
		<tr>
			<td>Streptomycin sulphate</td>
			<td>Sigma-Aldrich</td>
			<td>S9137</td>
			<td>/</td>
		</tr>
		<tr>
			<td>Styrofoam/nitrogen resistant box</td>
			<td>/</td>
			<td>/</td>
			<td>All boxes which can contain liquid nitrogen are ok, as long as the operator is comfortable. Kitazato box is called &#34;Cooling Rack&#34;</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma-Aldrich</td>
			<td>S1888</td>
			<td>/</td>
		</tr>
		<tr>
			<td>Timers</td>
			<td>/</td>
			<td>/</td>
			<td>All timers which can be set on times until 9 minutes are ok</td>
		</tr>
	</tbody>
</table>

minimum volume vitrification of immature feline oocytes

在野生动物保护计划中，游戏银行对于保护有价值的个体和稀有物种的遗传资源以及促进生物多样性保护至关重要。在幸福，大多数物种面临灭绝的威胁，国内品种被用作提高种质库协议效率的模型。在卵母细胞冷冻保存技术中，玻璃化在人类和兽医辅助生殖中越来越受欢迎。最初为人类卵母细胞和胚胎开发的冷冻体，已证明非常适合猫卵母细胞。该方法具有若干优点，如现场条件的可行性和工艺速度，当需要处理多个样品时，这都很有帮助。然而，效率在很大程度上取决于操作员的技能，需要实验室内和实验室间标准化以及人员培训。该协议描述了从卵母细胞收集到加热，商业支持上未成熟猫卵母细胞的最小体积玻璃化。按照协议，在变暖时保持卵母细胞的完整性和生存能力（高达90%）虽然气候变暖后成熟和胚胎发育结果仍有改进的余地，但可以预期。

根据目前的协议（图1和补充图1），猫卵母细胞玻璃化和变暖后，绝大多数的卵母体存活下来。玻璃化后，除其他技术外，在光学显微镜上可以评估其形态完整性22或使用重要污渍。后者之一是荧光素二甲酸酯/碘化钠（FDA/PI），它允许识别可行的（明亮的绿色荧光）和死细胞（红荧光）。图 2显示了在加热后被染有 FDA/PI ?...

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

Minimum Volume Vitrification of Immature Feline Oocytes

本手稿描述了一个协议，用于在商业支架上使用实验室制造的介质对未成熟的猫卵母细胞进行最小体积玻璃化。它涵盖了从卵母细胞分离从外体内性腺到玻璃化和变暖的每一步。

未成熟费林卵母细胞的最小体积玻璃化

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

该协议有助于通过称为玻璃化的低温保存技术长期保护猫卵母细胞，这对动物物种的生育和生物多样性保护至关重要。玻璃化的主要优点是速度，因为它可以在17分钟内完成，如果有液氮的话，在野外条件下可行。家猫是野生濒危猫科动物的可行模式。冷冻保存协议的开发和优化将允许为猫科动物保护计划建立游戏生物库。首先准备一个重现玻璃化板，一排有三个锥形井。在第一口井和三井中加入20微升平衡溶液，在第二和第三口井中加入300微升玻璃化溶液。要平衡积液卵母细胞复合物或 COC，请使用小孔移液器将一个或多个复合物转移到第一口井的底部。缓慢地将 20 微升的均衡溶液添加到跌落的边界中，等待三分钟，然后再添加 240 微升的均衡溶液，等待 9 分钟。在等待时，准备一个带液氮的盒子，并用实验或猫识别码标记玻璃化支持。将盒子和支撑放在立体显微镜附近。平衡 COC 后，在 90 秒内执行玻璃化。要对 COC 进行玻璃化，请用第二孔的玻璃化溶液填充小孔移液器。将 COC 从第一个井的底部移动，并移动到第二个井的表面。使用玻璃化溶液清洗移液器，然后将 COC 移动到井的另一个区域，然后混合周围的溶液。用从井的另一个区域填充移液器。移动 COC，将周围的介质与移液器混合。重复此过程一次。接下来，用第三井的玻璃化溶液清洗移液器，并用它来将COC移动到第三井的底部。再次，混合周围的解决方案。从井的另一个区域用溶液填充移液器后，移动 COC 并混合溶液。重复此过程。用井的另一个区域的玻璃化溶液填充移液器尖端，并用它来将COC加载到靠近尖端的玻璃化支撑条上，然后吸气多余的介质。立即将玻璃化支持液中浸在液氮中，并使用夹子关闭，确保保持浸入。将装载的玻璃化支架存放在杯中，并存放在储存液氮罐中，直到加热。将加热台靠近立体显微镜，将其打开至 38 摄氏度。加热舞台顶部的培养皿盖，然后将玻璃化支架从储罐转移到含有液氮的盒子里。准备一排重现板，用于加热每个玻璃化支撑。在第一口井中加入300微升稀释溶液，在第二和第三井中加入300微升的洗涤溶液。将解冻溶液从培养箱中取出，在培养皿的盖子上滴下100微升。使用夹子打开液氮内的玻璃化支撑。将 Petri 盘盖放在立体显微镜下，然后快速拿起玻璃化支撑，将其条带浸入落点中，将其移动，直到所有 COC 分离。一旦跌落为空，立即从跌落中卸下支撑，但将 COC 留在解冻溶液中一分钟。用稀释溶液填充移液器，并用它来将COC从解冻溶液滴到第一井的底部，使用稀释溶液。把 COC 留在那里三分钟。用洗涤液从第二个井清洗移液器，然后采取COC，并移动到第二个井的底部。等待五分钟。用第三个井的溶液清洗移液器，并用它来将 COC 从第二个井移动到第三井的表面。等待 COC 触摸第三个井的底部。然后用另一个井区重复此过程。完成后，用培养介质清洗移液器，将卵母细胞移动到培养皿中。根据该协议，绝大多数的游戏体在卵母细胞玻璃化和变暖后存活。此处显示了一个可行的维他化温暖猫积卵母细胞复合物与荧光素二甲酸酯和碘化二甲酸酯的代表性图片。下表显示了玻璃化后生存的百分比，其中描述了用于体外成熟的卵母细胞的加热后数据。在435个卵母细胞中，395个存活下来，在变暖后总体获得90.8%的生存能力。然而，一些形态的变化可以注意到后，变暖。最常见的形态异常是表粒形状和造粒的变化，部分或有时完全丧失积糖细胞，以及佐纳佩普图西达骨折。尝试此过程时，请提前准备介质，以便它们在使用前达到正确的温度，并遵循协议温度和时间以避免细胞毒性。该协议也可以适用于其他物种的卵母细胞。如果成功，这也可能有助于为濒危猫科动物的生物多样性保护提供细菌。

Research

JoVE Journal

Developmental Biology

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

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

荧光蛋白的表达<em&gt;披文昌鱼</em&gt;通过基因注射到未受精的卵母细胞

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

科研

发育生物学

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

基于核仁染色质组织分离鼠标窦卵母细胞与表征

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

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

修改MicroSecure玻璃化：一种安全，简便，高效的冷冻程序人胚泡

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

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

马卵母细胞染色体分离，组蛋白乙酰化和主轴形态分析

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

Generation of Genetically Modified Mice through the Microinjection of Oocytes

通过显微注射卵母细胞生成转基因小鼠

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

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

基于神经网络的发育能力或不称职小鼠全生长卵母细胞的识别

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

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

家兔模型最佳胚胎阶段微创胚胎移植与胚胎玻璃化

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

Human Blastocyst Biopsy and Vitrification

人类囊胚活检和细胞化

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

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

牛早期蚁卵泡卵母细胞的体外培养策略

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

Separation of Follicular Cells and Oocytes in Ovarian Follicles of Zebrafish

斑马鱼卵巢卵巢中卵泡细胞和卵母细胞的分离

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