Name: bottlenose dolphin (tursiops truncatus) spermatozoa: collection, cryopreservation, and heterologous in vitro fertilization
Uploaded: 2017-08-21T12:30:00.000+00:00
Duration: 11 min 42 s
Description: Watch this Scientific Journal Video about Bottlenose Dolphin Tursiops truncatus Spermatozoa: Collection, Cryopreservation, and Heterologous In Vitro Fertilization at JoVE.com

他的工作是由经济和竞争力 (AGL2015-70140-R 的 D.Rizos)、 AGL2015 66145R A.古铁雷斯阿丹和 J.F.· 佩雷斯 · 古铁雷斯和塞内卡穆尔西亚基金会 (格兰特 20040/胚芽/16 F.加西亚 · 巴斯克斯) 西班牙语部资助的。

伦理声明: 所有的实验程序的审查和核准由体制动物护理和使用委员会和研究国家研究所 y y 法规国家博览会 （老龄研究所）。所有实验了按照指南 》 的护理和使用实验室的动物，通过社会的再生产研究和海洋哺乳动物照顾动物福利法。
 1.海豚精子收集和冷冻保存 <ol><li>制备 <ol><li>使 FERT 介质 （肝素和亚硫磺酸免）。准备 FERT 人 TALP 培养基 25 毫米碳酸氢盐、 乳酸钠 22 毫米，1 毫米丙酮酸钠、 6-毫克/毫升无脂肪酸牛血清白蛋白，与 10 毫克/毫升肝素 (pH 7.4)。 
		注意: 使用日准备这种媒介和保持它在 38.5 ° C，5%CO 2，并在使用前至少 2 小时最大湿度与空气气氛下。</li>
		<li>准备三鸡蛋蛋黄基于缓冲区。溶解 30.28 g/L 三、 16.75 G-L 柠檬酸、 12.5 G-L 果糖 0.5 g/L 链霉素在纯水中的 (ph 值 7.3、 渗透压 = 310 收音机/公斤)。添加蛋黄 20%(v/v)。</li>
	</ol></li> <li>培训证明的育种男性的宽吻海豚躺在背 recumbence 附近游泳池边上，为自愿采精。 
	注: 雄性海豚应培训 6 个月以上的正面，如前面所述 4。</li>
	<li>通过轻轻按压在生殖器槽的海豚颅骨的区域，征求自愿挤压的阴茎从槽内生殖器执行各种触觉刺激。</li>
	<li>实现一个完整的勃起，直接后阴茎尖端到无菌容器中以获得射精。</li>
	<li>直到分析保持在 37 ° C 的精子样本。对于连续射精集合，每次使用新的丙烯玻璃重复步骤 1.3 和 1.4。</li>
	<li>集合后立即, 确定使用以前加热到 37 ° C.评价颜色、 ph 值和渗透压 （使用微压计） 射出精液的毕业的玻璃缸体积。</li>
	<li>评价在射精，精子的浓度将 10 微升的精子暂停添加到包含 90 µ L 的蒸馏水的离心管。确定使用计数室精子的精子浓度。 
	注: 评估期间，剩下的留射出精液在 37 ° C.</li>
	<li>动力参数评估。
	<ol><li>采取 10 微升的精子样本并将其放置在预热的精子会议厅。</li>
		<li>评价动力使用计算机辅助精子分析系统，以前验证的宽吻海豚，能客观地评估精子动力 2. 
		注: 如果有必要，样本的稀释精子 FERT 介质 （肝素和亚硫磺酸免） 与精子活力充分可视化。评估期间，维持在 37 ° C 样品加热的显微镜舞台上。</li>
	</ol></li> <li>20 微升的精子样本，并评价的可行性和精子形态作为先前描述 5。</li>
	<li>转移到离心管中精子样本。离心机在 250 x g 5 分钟精子样本。后确定采用计数室的精子浓度，调整到 400 x 10 6 精子/毫升精子浓度。 
	注意: 如果有必要，稀释与孤立精浆从同一射精过量体积从集中射精颗粒离心 （250 x g，10 分钟）。
	<ol><li>，当然 5 分钟，慢慢稀释精子样本 1:1 (v/v) 与含有 1.5%甘油三鸡蛋蛋黄基于缓冲区。放置包含精子悬浮在 5 ° C，1 小时 30 分钟的管</li>
		<li>执行第二次稀释的精子悬架的三蛋含有甘油获得 3%最后甘油浓度与终浓度为 200 x 10 6 精子/mL 的蛋黄基于缓冲区。保持精子悬浮在 5 ° C 10 分钟</li>
	</ol></li> <li>加载到 0.25 毫升吸管的精子悬浮。热封麦秆。在机架 4.5 厘米以上为 10 分钟的液态氮中稻草秸秆陷入液态氮和存储直到评价的地方。</li>
</ol> 2。异源 体外 受精使用透明完整牛卵母细胞 <ol><li>制备 <ol><li>准备染色溶液和安装介质。
		<ol><li>准备 Hoechst 股票溶液溶解 1 毫克的赫斯特在 1 毫升的蒸馏水。30 微升整除的数，保持他们在黑暗中在-20 ° C 直到使用。通过将 25 微升的赫司特公司股票解决方案添加到 5 毫升的磷酸盐缓冲液 (PBS) 辅以 1 g/L PVA 溶液染色。拌匀，保持在黑暗中在 4 ° C，直到使用。</li>
			<li>准备安装介质混合 6.25 毫升的 PBS 6.25 毫升的甘油和 6.25 微升的赫司特公司股票解决方案。拌匀，保持在黑暗中在 4 ° C，直到使用。</li>
		</ol></li> <li>介质准备卵母细胞的采集和 体外 受精。
		<ol><li>盐溶液法制备水中加入 0.5 毫升的庆大霉素对 0.9%氯化钠蒸馏。</li>
			<li>10 吴/mL 表皮生长因子 (EGF) 和 10%胎牛血清 (FCS) 加入 10 毫升的中医 199 准备股票成熟培养液。保持这两种媒体在 38.5 ° C，5%CO 2，并在使用前至少 2 小时最大湿度与空气气氛下。</li>
		</ol></li></ol></li> <li>收集在 33-35 ° C 的盐溶液中屠宰场卵巢，运到 4 小时内实验室从集合。</li>
	<li>一次在实验室里，清洗卵巢三次删除碎片和血液使用生理盐水以前加热到 37 ° C.保持卵巢在 37 ° C 的盐溶液中的过程。</li>
	<li>与 18 G 2 到 8 毫米蓇葖果抽吸针和 10 毫升注射器。收集 50 毫升管吸气的卵泡液。</li>
	<li>允许卵泡液含卵母细胞大约 15 分钟在 37 ° C，直到细胞沙站。删除与巴斯德吸管管上的清液和再次悬浮颗粒在 PBS。</li>
	<li>在 90 毫米菜在体视显微镜下恢复卵丘-卵母细胞复合体 (COCs)。</li>
	<li>洗 COCs 三次在 PBS 中和一次成熟培养液中。</li>
	<li>转让正常形态 COCs 4 井菜，每 500 毫升的培养液，每井 50 COCs 组含。 
	注意: 在这里，三个井 &#8211; 异源体外受精、 一个控制好进行同源试管受精和一只含一井成熟 Coc 作为一个孤雌生殖的控件 &#8211; 使用了。</li><li>孵育 24h 在 38.5 ° C，5%CO 2，最大湿度与空气气氛下 COCs。</li>
	<li>准备介质和密度梯度。
	<ol><li>准备受精介质 (FERT)，补充 25 毫米碳酸氢盐、 乳酸钠 22 毫米，1 毫米丙酮酸钠、 6 毫克/毫升无脂肪酸牛血清白蛋白，与 10 毫克/毫升肝素 FERT 人 TALP 介质。在 38.5 ° C，5%CO 2，并在使用前至少 2 小时最大湿度与空气气氛下维护。</li>
		<li>通过添加 1 毫升的密度梯度介质到 15 毫升管和 3 毫升的洗涤液 15 毫升管准备密度梯度。保持他们在至少 1 h.38.5 ° C</li>
	</ol></li> <li>洗 体外 成熟卵母细胞 FERT 介质中两次。</li>
	<li>转让，卵母细胞 4 井菜，每个含 50 COCs FERT 250 毫升。同时准备受精的精子保持 38.5 ° C，5%CO 2，并在与最高湿度空气气氛下的 4 井菜。</li>
	<li>解冻冷冻的秸秆含海豚精子在水浴在 37 ° C 为 50 s. 
	注: 牛精子进行同源试管受精必须被并行处理，对同源的牛体外受精后的标准协议。</li>
	<li>带 10 微升的精子样本，并将其置于预热的精子计数室。评估采用计算机辅助精子分析系统，以前验证的物种，能客观地评估精子活力的动力。在此期间，保持精子样本在 38.5 ° C.</li>
	<li>将精子样本添加到密度梯度管 （步骤 2.10.2）。离心机在 250 x g.10 分钟</li>
	<li>小心取出上清液使用巴斯德吸管。添加到包含颗粒管 3 毫升的洗涤液。再次通过轻轻搅拌悬浮颗粒。</li>
	<li>离心机在 250 x g.5 分钟删除与巴斯德吸管到 300 µ L 精子卷清。</li>
	<li>添加 5 µ L 的精子悬浮到离心含有 95 µ L 的蒸馏水。保持精子悬浮在 38.5 ° C.填充单元格计数室与 10 微升 1:20 的精子稀释和测量浓度。计算的金额必须添加到 100 μ L 的精子获得 2 x 10 6 精子/mL 的 FERT 介质。</li>
	<li>将计算的 FERT 和精子卷添加到 15 毫升管和轻轻地混用，巴斯德吸管。</li>
	<li>添加 250 µ L 的精子 FERT 悬浮每口井的含有 FERT 介质与成熟的卵母细胞获得终浓度为 1 × 10 6 精子/mL 4 井菜。</li>
	<li>共同孵育 18 小时在 38.5 ° C，5%CO 2，并在与最高湿度空气气氛下配子。</li>
	<li>配制培养基的基础合成的输卵管液和 5 %fcs 的补充。准备 25 微升的合成输卵管液的培养液滴在 35 毫米菜满 3 毫升的矿物油。在 38.5 ° C，5%CO 2，并在使用前至少 2 小时最大湿度与空气气氛下维护。</li>
	<li>在 2.5 h 后共同孵育，从盘子里删除 20 卵母细胞; 保持在相同的条件下孵化器中剩余的卵母细胞。
	<ol><li>通过大力用一半的卵母细胞通过窄口径巴斯德吸管移液器 10 倍删除松散地连接的精子。</li>
		<li>修复 20 的卵母细胞体外 50 µ L 的 0.5%的戊二醛 30 分钟洗 5 分钟 PBS 卵母细胞转移到 50 µ L 的赫斯特染液 15 分钟的卵母细胞和洗卵母细胞体外 PBS 为 5 分钟</li>
		<li>单独将卵母细胞转移到 2 µ L 液滴的安装介质在每张幻灯片 10 卵率。轻轻地盖上盖玻片并加盖指甲油。</li>
		<li>的附加到使用配有蓝光光学和 361 nm 励磁筛选器在放大 40 倍相衬显微镜的牛卵母细胞的精子计数。</li>
	</ol></li> <li>共同培养 12 小时后, 删除 10 推定受精卵从 4 井菜; 保持剩余的推定受精卵在相同的条件下孵化。
	<ol><li>转让 10 推定受精卵到 15 毫升离心管 PBS 和涡流轻轻为 2 分钟，去除卵丘细胞含有 2 毫升。洗两次在 PBS，修复和污渍，如上文所述 （第 2.23.2 步。）</li>
	</ol></li> <li>18，20，22 和共同培养 24 小时删除 10 后推定受精卵从 4 井菜和涡，修复，和玷污它们如上文所述 （步骤 2.23.2）。保持其余的在相同的条件下孵化受精卵。
	<ol><li>Hoechst 染色评价原核形成和在同源和异源体外受精群体相-对比度/蓝光显微镜下的多精受精。</li>
		<li>评估在每个采样 10 随机选择推定混合受精卵原核形成。确认下共焦激光扫描显微镜在 488 nm 氩激光激发和 515 到 530 nm 检测原核形成。</li>
		<li>光节推定合子按顺序 （2 毫米） 和采集图像使用图像处理软件。可视化使用 3D 分析软件包。</li>
		<li>后共同培养 24 小时，洗 50 推定合子四次在 PBS 中，两次在培养基。</li>
	</ol></li> <li>他们 25 组移交 microdroplets 培养基以前编写和平衡。</li>
	<li>维护在 38.5 ° C 下大气的 5%O 2 / 5%CO 2 中与最大湿度空气。</li>
	<li>后 26 和 28 h 的共同孵育，从菜和涡、 修复、 删除 10 推定合子和玷污它们，如上文所述 （步骤 2.23.2）。</li>
	<li>后的文化的 48 h，观察在体视显微镜下的劈。</li>
	<li>通过将胚胎转移到 5 毫克/毫升蛋白酶解卸下卵透明带 (ZP)。</li>
	<li>三次单独在 PBS 洗每个胚胎。管理单元-冻结他们在 0.2 毫升的微量离心管中的液态氮内。在-80 ° C 保存它们，直到分析。</li>
</ol> 3。聚合酶链反应 <ol><li>材料和试剂 注: 试剂、 材料和设备用于执行 PCR 协议详细材料表和包括 PCR 管架和微 （P2，P20，P200，P1000），一套 1.5 毫升微量离心管、 PCR 管帽、 热循环、 紫外线照射、 琼脂糖凝胶和缓冲区 (三硼酸盐乙二胺四乙酸 (TBE))。
	<ol><li>轻轻地解冻所有试剂在冰上的。在整个试验冰保持他们。使用 deoxynucleotides (dNTPs; dATP，dCTP，dTTP 和 dGTP)，目标引物对海豚 18 S 核糖体蛋白 (DQ404537.1); 参考基因编码R1: TTGGACACACCCACGGTGCGG 和 F2: CAGAAGGACGTGAAGGATGGA)，DNA 聚合酶、 DNA 聚合酶、 DNA 模板、 无菌水和氯化镁 (氯化镁 2 在终浓度为 5.0 m m) 5 X 缓冲区。</li>
		<li>琼脂糖凝胶和样品制备。
		<ol><li>准备海豚和牛精子 DNA 样本。
			<ol><li>解冻在 37 ° C 的水浴中每一物种冷冻秸秆 50 美国加入 3ml 的洗涤液。离心机在 250 x g 10 分钟，除去上清。添加 30 微升国有贸易企业裂解缓冲液 （50 毫米三-盐酸，ph 值 8; 20 毫米氯化钠; 和 1 毫米 0.1 %sds） 2 微升蛋白酶 K （20 毫克/毫升） 和 5 µ L 的醇 (DTT);最终浓度: 150 毫米。一夜之间保持在 55 ° C.添加 200 µ L 的超纯水。离心机在 250 x g 5 分钟保持上清液。DNA 浓度用分光光度计测量。</li>
			</ol></li> <li>从海豚精子 (即， 40、 4 和 0.4 吴/mL) 用超纯水检查 PCR 条件有能力执行串行稀释度的 DNA检测少量的海豚 DNA。</li>
			<li>执行牛精子 DNA (即 4,000、 400、 40 和 4 吴/mL) 用超纯水检查 PCR 条件是能够检测少量的牛 DNA 系列稀释。</li>
			<li>编写的海豚和牛的胚胎。解冻胚胎在冰上。添加 8 µ L 的 100 微克/毫升蛋白酶 K 和维护一夜之间在 55 ° c。若要停止反应，请将样品放在 96 ° C 为 5 分钟</li>
			<li>准备 2%琼脂糖凝胶电泳 TBE 缓冲液和添加的核酸染色使用标准技术 20 微升。</li>
		</ol></li> <li>聚合酶链反应制备。
		<ol><li>标签 PCR tube(s): 海豚系列稀释 (40、 4 和 0.4 吴/mL)、 牛系列稀释 （4,000、 400、 40 和 4ml 吴），同源牛的两个细胞的胚胎、 两细胞推定的外源胚胎和阴性对照。</li>
			<li>编写一个主的混合物，在无菌 1.5 毫升离心管中给每个反应 25 微升最终卷。
			<ol><li>添加 5 微升 5 X DNA 聚合酶弹性缓冲，0.5 微升的 dNTPs （10 毫米） 1.5 µ L 的氯化镁 2 （25 毫米），0.5 µ L 的正向和反向引物 (10 µ M)，1.5 微升的 DNA 模板 （精子） 或 8 µ L 的 DNA 模板 （2-细胞胚胎）由 25 微升最后一卷的 0.07 µ L Taq 聚合酶和无菌蒸馏水。拌匀。</li>
			</ol></li></ol></li></ol></li> <li>扩增 <ol><li>管置于热循环。选择以下程序: 94 ° C 3 分钟;94 ° C 为 15 的 40 个周期 25 59 ° C s s，72 ° C 为 20 s;和 72 ° C 为 5 分钟启动程序。</li>
		<li>存储在 4 ° C.添加 2.5 微升的加载到每个 PCR 产物在 4 ° C 和负载 15 微升混合物的缓冲区到 2%琼脂糖凝胶的井管。一个 DNA 梯标记用于 PCR 片段的准确大小估计。</li>
		<li>执行电泳 15 分钟，使 DNA 迁移。可视化乐队使用 UV 光源。</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Montano, G. 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=Evaluation+of+motility,+membrane+status+and+DNA+integrity+of+frozen-thawed+bottlenose+dolphin+(Tursiops+truncatus)+spermatozoa+after+sex-sorting+and+recryopreservation.">Evaluation of motility, membrane status and DNA integrity of frozen-thawed bottlenose dolphin (Tursiops truncatus) spermatozoa after sex-sorting and recryopreservation.</a> Reproduction. 143 (6), 799-813 (2012).</li><li>S&#225;nchez-calabuig, M. J., 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=Validation+of+a+field+based+chromatin+dispersion+assay+to+assess+sperm+DNA+fragmentation+in+the+bottlenose+dolphin+(Tursiops+truncatus).">Validation of a field based chromatin dispersion assay to assess sperm DNA fragmentation in the bottlenose dolphin (Tursiops truncatus).</a> Reprod Domest Anim. 49 (5), 761-768 (2014).</li><li>S&#225;nchez-calabuig, M. J., 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=Heterologous+murine+and+bovine+IVF+using+bottlenose+dolphin+(Tursiops+truncatus)+spermatozoa.">Heterologous murine and bovine IVF using bottlenose dolphin (Tursiops truncatus) spermatozoa.</a> Theriogenology. 84 (6), 983-994 (2015).</li><li>Keller, K. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Training+of+the+Atlantic+bottlenose+dolphins+(Tursiops+truncatus)+for+artificial+insemination.">Training of the Atlantic bottlenose dolphins (Tursiops truncatus) for artificial insemination.</a> IAAAM. 14 (22), 22-24 (1986).</li><li>Robeck, T. R., O'brien, 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=Effect+of+cryopreservation+methods+and+precryopreservation+storage+on+bottlenose+dolphin+(Tursiops+truncatus)+spermatozoa.">Effect of cryopreservation methods and precryopreservation storage on bottlenose dolphin (Tursiops truncatus) spermatozoa.</a> Biol Reprod. 70 (5), 1340-1348 (2004).</li><li>Coy, P., 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=Hardening+of+the+zona+pellucida+of+unfertilized+eggs+can+reduce+polyspermic+fertilization+in+the+pig+and+cow.">Hardening of the zona pellucida of unfertilized eggs can reduce polyspermic fertilization in the pig and cow.</a> Reproduction. 135 (1), 19-27 (2008).</li><li>Seager, S., W, G., Moore, L., Platz, C., Kirby, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Semen+collection+(electroejaculation),+evaluation+and+freezing+in+the+Atlantic+bottlenose+dolphin+(Tursiops+truncatus).">Semen collection (electroejaculation), evaluation and freezing in the Atlantic bottlenose dolphin (Tursiops truncatus).</a> Proceedings of the American Association of Zoo Veterinarians. 136, (1981).</li><li>Schroeder, J. P., Leatherwood, S., Reeves, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Breeding+Bottlenose+Dolphins+in+captivity.">Breeding Bottlenose Dolphins in captivity.</a> The Bottlenose dolphin. , 447-460 (1990).</li><li>S&#225;nchez-Calabuig, M. J., 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+cryopreservation+on+the+sperm+DNA+fragmentation+dynamics+of+the+bottlenose+dolphin+(Tursiops+truncatus).">Effect of cryopreservation on the sperm DNA fragmentation dynamics of the bottlenose dolphin (Tursiops truncatus).</a> Reprod Domest Anim. 50 (2), 227-235 (2015).</li><li>Robeck, T. R., 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=Estrous+cycle+characterisation+and+artificial+insemination+using+frozen-thawed+spermatozoa+in+the+bottlenose+dolphin+(Tursiops+truncatus).">Estrous cycle characterisation and artificial insemination using frozen-thawed spermatozoa in the bottlenose dolphin (Tursiops truncatus).</a> Reproduction. 129 (5), 659-674 (2005).</li><li>Robeck, T. R., 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=Development+and+evaluation+of+deep+intra-uterine+artificial+insemination+using+cryopreserved+sexed+spermatozoa+in+bottlenose+dolphins+(Tursiops+truncatus).">Development and evaluation of deep intra-uterine artificial insemination using cryopreserved sexed spermatozoa in bottlenose dolphins (Tursiops truncatus).</a> Anim Reprod Sci. 139 (1-4), 168-181 (2013).</li><li>Schroeder, J. P., Leatherwood, S., Reeves, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Breeding+Bottlenose+Dolphins+in+captivity.">Breeding Bottlenose Dolphins in captivity.</a> The Bottlenose dolphin. , 447-460 (1990).</li><li>Madeddu, 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+cooling+rate+on+the+survival+of+cryopreserved+rooster+sperm:+Comparison+of+different+distances+in+the+vapor+above+the+surface+of+the+liquid+nitrogen.">Effect of cooling rate on the survival of cryopreserved rooster sperm: Comparison of different distances in the vapor above the surface of the liquid nitrogen.</a> Anim Reprod Sci. , (2016).</li></ol>

作者没有透露。

对于许多不同的哺乳类动物，有多样的优势，以利用冷冻精子。这些包括转移宝贵的遗传材料、 潜在的全球分布、 污染、 低风险和几十年来保持雄性配子的能力的能力。利用冷冻精子的宽吻海豚，至关重要，因为这一物种保护下附录 II 的引证，限制的运输和交换的动物之间不同的水上公园。海豚运输是创建后勤问题和危险的活动。然而，避免运送他们也有的后果，例如引入到动物圈养种群的血...

辅助生殖技术在野生动物，包括海洋哺乳动物发育不良。缺乏敏感的方法，以评估精子受精成功贡献的物种，如海豚生殖技术发展缓慢。它不是直到最近的宽吻海豚 (海豚截形) 基本精参数是报告的1，2。但是，变量，如运动和形态，虽然广泛应用，给生殖效率的资料有限。精子质量的最佳指标是施肥潜力的评价。
最近，我们小组用一种方法来评估海豚精子通过评估雄原核形成和/或混合胚胎形成异源体外受精使用透明完整牛卵母细胞3后的施肥的潜力。海豚-牛异体受精使用作为它克服困难获得海豚卵母细胞的重要优点同源的体外受精，并有助于行之有效体外牛卵母细胞成熟系统的使用。为了避免种属特异性，异体受精通常被执行不到位的 ZP。虽然它允许对精子顶体反应的精子与卵黄膜融合的能力的评价，它损害的评价与施肥相关的其他功能。所述的过程使用透明完整卵母细胞和允许下列参数评价: 精子卵具约束力和附件、 渗透、 多精受精、 原核形成和混合胚胎卵裂。
在这里，我们目前为精子集合、 基本精子分析、 精子冷冻的海豚精子功能评价的几种协议通过评估雄性原核和/或混合胚胎的形成异源体外受精后使用透明完整牛卵母细胞。

<table><tbody><tr><td>FERT-TALP medium</td><td>Merck</td><td></td><td></td></tr><tr><td>TCM-199</td><td>Sigma</td><td>M-4530</td><td></td></tr><tr><td>Hoechst 33342</td><td>Sigma</td><td>B-2261</td><td></td></tr><tr><td>4-well dishes</td><td>Nunc</td><td>176740</td><td></td></tr><tr><td>Density gradient BoviPure</td><td>Nidacon International</td><td>BP-100</td><td></td></tr><tr><td>Washing solution Boviwash</td><td>Nidacon International</td><td>BW-100</td><td></td></tr><tr><td>Magnesium chloride</td><td>Promega</td><td>A35 1H</td><td></td></tr><tr><td>Sterile water</td><td>Mili Q sintesis A10 Millipore</td><td>A35 1H</td><td></td></tr><tr><td>Buffer Tris Borate EDTA</td><td>Sigma</td><td>T4415</td><td></td></tr><tr><td>MB agarose</td><td>Biotools</td><td>20.012</td><td></td></tr><tr><td>5X GoTaq flexi buffer</td><td>Mili Q sintesis A10 Millipore</td><td>M 890 A</td><td></td></tr><tr><td>MB agarose</td><td>Biotools</td><td>20.012</td><td></td></tr><tr><td>Taq polymerase</td><td>Promega</td><td></td><td></td></tr><tr><td>SafeView</td><td>NBS Biologicals Ltd.</td><td>M 890 A</td><td></td></tr><tr><td>Makler counting chamber</td><td>Sefi Medical</td><td></td><td></td></tr><tr><td>Thoma chamber</td><td>Hecht-Assistant</td><td></td><td></td></tr><tr><td>pHmeter MicropH 2000</td><td>Crison Instruments</td><td></td><td></td></tr><tr><td>Osmometer Advanced micro osmometer 3300</td><td>Norwood</td><td></td><td></td></tr><tr><td>Computer assisted sperm analysis system</td><td>Projectes y Serveis R+D</td><td></td><td></td></tr><tr><td>Stereomicroscope MZ 95</td><td>Leica</td><td></td><td></td></tr><tr><td>Epifluorescent optics Eclipse Te300</td><td>Nikon</td><td></td><td></td></tr><tr><td>Confocal assistant 4.02 software</td><td>Bio-Rad</td><td>3D analysis software</td><td></td></tr><tr><td>Confocal laser scanning microscopy</td><td>Bio-Rad</td><td></td><td></td></tr><tr><td>Micropippetes (P2. P20, P200, P1000)</td><td>Gilson</td><td></td><td></td></tr><tr><td>Microcentrifuge tubes</td><td>VWR</td><td></td><td></td></tr><tr><td>UV iluminator</td><td>Bio-Rad</td><td></td><td></td></tr><tr><td>PCR Thermal cycler Primus 96 Plus</td><td>MWG AG Biotech</td><td></td><td></td></tr></tbody></table>

bottlenose dolphin (tursiops truncatus) spermatozoa: collection, cryopreservation, and heterologous in vitro fertilization

深低温保存的海豚精子的使用促进水生公园之间的遗传材料的交换，并使精子可到实验室进行研究，以进一步加深对海洋哺乳动物繁殖。外源体外受精，更换为异体受精，可以提供一种手段来测试精子生育潜力;研究生理学配子和胚胎的早期发育;和避免使用宝贵的海豚卵，很难获得。在这里，我们目前已成功地用于收集和海豚精子冷冻保存的协议。集合的精子进行人工刺激对经过训练的海豚。超低温保存是甘油使用三蛋黄基于扩展程序完成的。另外，我们提出一种协议，它描述使用海豚精子和牛卵母细胞的异种受精和用于验证所产生的胚胎，应用 pcr 技术的混合性质。异体受精提出了施肥的问题，可以作为一种工具用于研究配子生理学和早期胚胎发育。此外，异体受精成功演示这种技术的潜力，来测试海豚精子受精能力，值得进一步的检查。

所有结果，表和在这里提出的数字 （1 和 2） 被都转载权限3。
海豚精子有高动力后冻结和解冻
显示百分比 （%± SD） 84.5 ± 5.3 共能动和 69.1 ± 5.1 渐进式活动精子冷冻海豚射精。在动力方面，这些数字是代表高质量深低温保存的精子。
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Watch this Scientific Journal Video about Bottlenose Dolphin Tursiops truncatus Spermatozoa: Collection, Cryopreservation, and Heterologous In Vitro Fertilization at JoVE.com

Bottlenose Dolphin Tursiops truncatus Spermatozoa: Collection, Cryopreservation, and Heterologous In Vitro Fertilization

在这里，我们目前成功地用于海豚精子收集、 冷冻保存和异源使用牛卵母细胞的体外受精效果的协议。

宽吻海豚 (海豚截形) 精子: 收集、 冷冻保存和外源体外受精

The use of cryopreserved dolphin spermatozoa facilitates the exchange of genetic material between aquatic parks and makes spermatozoa accessible to ...

bottlenose-dolphin-tursiops-truncatus-spermatozoa-collection

Research

JoVE Journal

Developmental Biology

Bottlenose Dolphin (Tursiops truncatus) Spermatozoa: Collection, Cryopreservation, and Heterologous In Vitro Fertilization

13.2K 次观看.  Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA). 在这里，我们目前成功地用于海豚精子收集、 冷冻保存和异源使用牛卵母细胞的体外受精效果的协议。

视频: Bottlenose Dolphin Tursiops truncatus Spermatozoa: Collection, Cryopreservation, and Heterologous In Vitro Fertilization

Isolation and Cryopreservation of Neonatal Rat Cardiomyocytes

Cell culture has become increasingly important in cardiac research, but due to the limited proliferation of cardiomyocytes, culturing cardiomyocytes is difficult and time consuming. The most commonly used cells are neonatal rat cardiomyocytes (NRCMs), which require isolation every time cells are needed. The birth of the rats can be unpredictable. Cryopreservation is proposed to allow for cells to be stored until needed, yet freezing/thawing methods for primary cardiomyocytes are challenging due to the sensitivity of the cells. Using the proper cryoprotectant, dimethyl sulfoxide (DMSO), cryopreservation was achieved. By slowly extracting the DMSO while thawing the cells, cultures were obtained with viable NRCMs. NRCM phenotype was verified using immunocytochemistry staining for &#945;-sarcomeric actinin. In addition, cells also showed spontaneous contraction after several days in culture. Cell viability after thawing was acceptable at 40-60%. In spite of this, the methods outlined allow one to easily cryopreserve and thaw NRCMs. This gives researchers a greater amount of flexibility in planning experiments as well as reducing the use of animals.

The isolation of neonatal rat cardiomyocytes is a time consuming and unpredictable procedure. This study describes methods for cryopreservation and thawing of neonatal rat cardiomyocytes that allows for more efficient use of cells. The thawed NRCMs can be used for various experiments without the need for performing isolations each time.

隔离和乳鼠心肌细胞冷冻保存

Cell culture has become increasingly important in cardiac research, but due to the limited proliferation of cardiomyocytes, culturing cardiomyocytes is ...

科研

发育生物学

In Vitro and In Vivo Models to Study Corneal Endothelial-mesenchymal Transition

Corneal endothelial cells (CECs) play a crucial role in maintaining corneal clarity through active pumping. A reduced CEC count may lead to corneal edema and diminished visual acuity. However, human CECs are prone to compromised proliferative potential. Furthermore, stimulation of cell growth is often complicated by gradual endothelial-mesenchymal transition (EnMT). Therefore, understanding the mechanism of EnMT is necessary for facilitating the regeneration of CECs with competent function. In this study, we prepared a primary culture of bovine CECs by peeling the CECs with Descemet's membrane from the corneal button and demonstrated that bovine CECs exhibited the EnMT process, including phenotypic change, nuclear translocation of &#946;-catenin, and EMT regulators snail and slug, in the in vitro culture. Furthermore, we used a rat corneal endothelium cryoinjury model to demonstrate the EnMT process in vivo. Collectively, the in vitro primary culture of bovine CECs and in vivo rat corneal endothelium cryoinjury models offers useful platforms for investigating the mechanism of EnMT.

In Vitro and In Vivo Models to Study Corneal Endothelial-mesenchymal Transition

A primary culture of bovine corneal endothelial cells was used to investigate the mechanism of corneal endothelial-mesenchymal transition. Furthermore, a rat corneal endothelium cryoinjury model was used to demonstrate corneal endothelial-mesenchymal transition in vivo.

在体外和体内模型来研究角膜内皮-间充质转换

Corneal endothelial cells (CECs) play a crucial role in maintaining corneal clarity through active pumping. A reduced CEC count may lead to corneal edema ...

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

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

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

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 ...

Collection of Serum- and Feeder-free Mouse Embryonic Stem Cell-conditioned Medium for a Cell-free Approach

The capacity of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) to generate various cell types has opened new avenues in the field of regenerative medicine. However, despite their benefits, the tumorigenic potential of ESCs and iPSCs has long been a barrier for clinical applications. Interestingly, it has been shown that ESCs produce several soluble factors that can promote tissue regeneration and delay cellular aging, suggesting that ESCs and iPSCs can also be utilized as a cell-free intervention method. Therefore, the method for harvesting mouse embryonic stem cell (mESC)-conditioned medium (mESC-CM) with minimal contamination of serum components (fetal bovine serum, FBS) and feeder cells (mouse embryonic fibroblasts, MEFs) has been highly demanded. Here, the present study demonstrates an optimized method for the collection of mESC-CM under serum- and feeder-free conditions and for the characterization of mESC-CM using senescence-associated multiple readouts. This protocol will provide a method to collect pure mESC-specific secretory factors without serum and feeder contamination.

This protocol provides a method for the collection of mouse embryonic stem cell (mESC)-conditioned medium (mESC-CM) derived from serum (fetal bovine serum, FBS)- and feeder (mouse embryonic fibroblasts, MEFs)-free conditions for a cell-free approach. It may be applicable for the treatment of aging and aging-associated diseases.

和无血清收集无饲养小鼠胚胎干细胞条件培养基的无细胞方法

The capacity of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) to generate various cell types has opened new avenues in the field ...

Evaluation of Intracellular Location of Reactive Oxygen Species in Solea Senegalensis Spermatozoa

Oxidative stress is one of the important factors in decreasing sperm quality. Developing efficient protocols for detecting reactive oxygen species (ROS) in spermatozoa is of high importance in any species, but these methods are rarely used and even less in teleost. Cryopreservation is a useful technique in aquaculture for different purposes, including gene banking and guaranteed sperm availability throughout the year. Freezing/thawing procedures could cause ROS production and damage the sperm cells. Considering the prospective damage that an excess of ROS production could cause in spermatozoa depending on their localization, here a detailed methodology to detect H2O2 and to evaluate its intracellular localization by confocal microscopy is provided. For this purpose, a combination of 3 fluorochromes (2&#8242;,7&#8242;-Dichlorodihydrofluorescein diacetate (DCFH-DA), a live mitochondria stain and 4&#8242;,6-Diamidino-2-phenylindole dihydrochloride (DAPI)) are used to evaluate the co-localization of H2O2 with spermatozoa nuclei or mitochondria in Solea senegalesis sperm samples.

Evaluation of Intracellular Location of Reactive Oxygen Species in Solea Senegalensis Spermatozoa

This protocol describes a detailed methodology for detecting H2O2 localization within Solea senegalensis spermatozoa using a sensitive fluorochrome DCFH-DA for ROS, a live mitochondria stain for mitochondria, and DAPI for nuclei visualization, respectively. The protocol is designed to be performed within 2 h with either fresh or thawed spermatozoa.

Solea Senegalensis精子中活性氧的细胞内定位评价

Oxidative stress is one of the important factors in decreasing sperm quality. Developing efficient protocols for detecting reactive oxygen species (ROS) ...

A High-Throughput Method For Zebrafish Sperm Cryopreservation and In Vitro Fertilization

This is a method for zebrafish sperm cryopreservation that is an adaptation of the Harvey method (Harvey et al., 1982).  We have introduced two changes to the original protocol that both streamline the procedure and increase sample uniformity.  First, we normalize all sperm volumes using freezing media that does not contain the cryoprotectant. Second, cryopreserved sperm are stored in cryovials instead of capillary tubes.  The rates of sperm freezing and thawing (&delta;&deg;C/time) are probably the two most critical variables to control in this procedure.  For this reason, do not substitute different tubes for those specified.  Working in teams of 2 it is possible to freeze the sperm of 100 males per team in ~2 hrs.  Sperm cryopreserved using this protocol has an average of 25% fertility (measured as the number of viable embryos generated in an in vitro fertilization divided by the total number of eggs fertilized) and this percent fertility is stable over many years.

A High-Throughput Method For Zebrafish Sperm Cryopreservation and In Vitro Fertilization

This is a high-throughput sperm cryopreservation protocol for zebrafish. Sperm cryopreserved using this protocol has an average of 25% fertility in subsequent vitro fertilization and is stable over many years.

对于斑马鱼精子超低温冷冻保存的一个高通量的方法和<em在体外施肥

This is a method for zebrafish sperm cryopreservation that is an adaptation of the Harvey method (Harvey et al., 1982).  We have introduced two changes to ...

生物学

Mouse Sperm Cryopreservation and Recovery using the I&middot;Cryo Kit

Thousands of new genetically modified (GM) strains of mice have been created since the advent of transgenesis and knockout technologies. Many of these valuable animals exist only as live animals, with no backup plan in case of emergency. Cryopreservation of embryos can provide this backup, but is costly, can be a lengthy procedure, and generally requires a large number of animals for success. Since the discovery that mouse sperm can be successfully cryopreserved with a basic cryoprotective agent (CPA) consisting of 18&#37; raffinose and 3&#37; skim milk, sperm cryopreservation has become an acceptable and cost-effective procedure for archiving, distributing and recovery of these valuable strains.
 Here we demonstrate a newly developed I&bull;Cryo kit for mouse sperm cryopreservation. Sperm from five commonly-used strains of inbred mice were frozen using this kit and then recovered. Higher protection ratios of sperm motility (&gt; 60&#37;) and rapid progressive motility (&gt; 45&#37;) compared to the control (basic CPA) were seen for sperm frozen with this kit in 5 inbred mouse strains. Two cell stage embryo development after IVF with the recovered sperm was improved consistently in all 5 mouse strains examined. Over a 1.5 year period, 49 GM mouse lines were archived by sperm cryopreservation with the I&bull;Cryo kit and later recovered by IVF.

Here we demonstrate the newly developed I&#x2022;Cryo kit for mouse sperm cryopreservation. Two-cell stage embryo development with frozen-thawed sperm was improved consistently in 5 mouse strains with the use of this kit. Over a 1.5 year period, 49 genetically modified mouse lines were archived by sperm cryopreservation with the I&#x2022;Cryo kit and later successfully recovered by IVF.

精子冷冻保存和恢复使用I ·冷冻包

Thousands of new genetically modified (GM) strains of mice have been created since the advent of transgenesis and knockout technologies.  Many of these ...

Cryopreservation of Zebrafish Spermatogonia by Whole Testes Needle Immersed Ultra-Rapid Cooling

Current trends in science and biotechnology lead to creation of thousands of new lines in model organisms thereby leading to the necessity for new methods for safe storage of genetic resources beyond the common practices of keeping breeding colonies. The main purpose of this study was to adapt the needle immersed vitrification (NIV) procedure to cryopreserve whole zebrafish testes. Cryopreservation of early-stage germ cells by whole testes NIV offers possibilities for the storage of zebrafish genetic resources, especially since after transplantation they can mature into both male and female gametes. Testes were excised, pinned on an acupuncture needle, equilibrated in two cryoprotective media (equilibration solution containing 1.5 M methanol and 1.5 M propylene glycol; and vitrification solution containing 3 M dimethyl sulfoxide and 3 M propylene glycol) and plunged into liquid nitrogen. Samples were warmed in a series of three consequent warming solutions. The main advantages of this technique are (1) the lack of spermatozoa after digestion of warmed testes thus facilitating downstream manipulations; (2) ultra-rapid cooling enabling the optimal exposure of tissues to liquid nitrogen therefore maximizing the cooling and reducing the required concentration of cryoprotectants, thereby reducing their toxicity; (3) synchronous exposure of several testes to cryoprotectants and liquid nitrogen; and (4) repeatability demonstrated by obtaining viability of above 50% in five different zebrafish strains.

The main purpose of this study was to adapt the needle immersed vitrification (NIV) procedure to cryopreserve whole zebrafish testes. Additionally, the repeatability of the method in five different zebrafish strains was tested.

全睾丸针浸泡超快速冷却法超低温保存斑马鱼细胞

Current trends in science and biotechnology lead to creation of thousands of new lines in model organisms thereby leading to the necessity for new methods ...

Fish Sperm Assessment Using Software and Cooling Devices

For gamete quality evaluation, there are innovative, rapid, and quantitative techniques that can provide useful data for aquaculture. Computerized systems for sperm analysis were developed to measure several parameters and one of the most commonly measured is the sperm motility.
Initially, this computer technology was designed for mammalian species, although it can also be used for fish sperm analysis. Fish have specific features that can affect sperm assessment such as a short motility time after activation and, in some cases, adaptation to lower temperatures. Thus, it is necessary to modify both software and hardware components to make motility analysis more efficient for fish sperm analysis. For mammalian sperm, the heating plate is used to maintain optimal temperatures of spermatozoa. However, for some fish species, it is advantageous to use a lower temperature to prolong the duration of motility, since the sperm remain active for less than 2 min. Therefore, cooling devices are necessary to refrigerate samples at constant temperature over the time of analysis, including on the optical microscope. This protocol describes the analysis of fish sperm motility using software for sperm analysis and new cooling devices to optimize the results.

The present protocol describes a procedure of fish sperm assessment using computer-assisted sperm analysis and cooling devices. The software gives a rapid, accurate and quantitative analysis of fish sperm quality based on spermatozoa motility, which can be a useful tool in aquaculture to improve reproduction success.

使用软件和冷却装置进行鱼类精子评估

For gamete quality evaluation, there are innovative, rapid, and quantitative techniques that can provide useful data for aquaculture. Computerized systems ...

<meta charset="utf8"></head><body>优化受威胁珊瑚物种的珊瑚精子冷冻保存

A Semi-Automated Workflow for the Cryopreservation of Coral Sperm to Support Biobanking and Aquaculture

Coral reefs are facing a crisis as the frequency of bleaching events caused by ocean warming increases, resulting in the death of corals on reefs around the world. The subsequent loss of genetic diversity and biodiversity can diminish the ability of coral to adapt to the changing climate, so efforts to preserve existing diversity are essential to maximize the resources available for reef restoration now and in the future. The most effective approach to secure genetics long-term is cryopreservation and biobanking, which permits the frozen storage of living samples at cryogenic temperatures in liquid nitrogen indefinitely. Cryopreservation of coral sperm has been possible since 2012, but the seasonal nature of coral reproduction means that biobanking activities are restricted to just a few nights per year when spawning occurs. Improving the efficiency of coral sperm processing and cryopreservation workflows is therefore essential to maximizing these limited biobanking opportunities. To this end, we set out to optimize cryopreservation processing pathways for coral sperm by building on existing technologies and creating a semi-automated approach to streamline the assessment, handling, and cryopreservation of coral sperm. The process, which combines computer-assisted sperm analysis, barcoded cryovials, and a series of linked auto-datasheets for simultaneous editing by multiple users, improves the efficiency of both sample processing and metadata management in the field. Through integration with cross-cutting research programs such as the Reef Restoration and Adaptation Program in Australia, cryopreservation can play a crucial role in large-scale reef restoration programs by facilitating the genetic management of aquaculture populations, supporting research to enhance thermal tolerance, and preventing the extinction of coral species. The described procedures will be utilized for coral cryopreservation and biobanking practitioners on reefs worldwide and will provide a model for the transition of cryopreservation technologies from research laboratories to large-scale applications.

<meta charset="utf8"></head><body>作者聚焦:为全球珊瑚礁恢复开发高效的低温保存和生物样本库技术

This protocol describes a semi-automated pathway to improve the efficiency and capacity of processing and cryopreservation of sperm from threatened coral species, aiming to secure genetic diversity and support reef restoration efforts.

用于珊瑚精子冷冻保存以支持生物样本库和水产养殖的半自动化工作流程

Coral reefs are facing a crisis as the frequency of bleaching events caused by ocean warming increases, resulting in the death of corals on reefs around ...

宽吻海豚 (海豚截形) 精子: 收集、冷冻保存和外源体外受精

摘要

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此视频中的章节

隔离和乳鼠心肌细胞冷冻保存

在体外和体内模型来研究角膜内皮-间充质转换

修改MicroSecure玻璃化:一种安全，简便，高效的冷冻程序人胚泡

和无血清收集无饲养小鼠胚胎干细胞条件培养基的无细胞方法

Solea Senegalensis精子中活性氧的细胞内定位评价

对于斑马鱼精子超低温冷冻保存的一个高通量的方法和施肥

精子冷冻保存和恢复使用I ·冷冻包

全睾丸针浸泡超快速冷却法超低温保存斑马鱼细胞

使用软件和冷却装置进行鱼类精子评估

用于珊瑚精子冷冻保存以支持生物样本库和水产养殖的半自动化工作流程

宽吻海豚 (海豚截形) 精子: 收集、 冷冻保存和外源体外受精

摘要

探索更多视频

此视频中的章节

隔离和乳鼠心肌细胞冷冻保存

在体外和体内模型来研究角膜内皮-间充质转换

修改MicroSecure玻璃化:一种安全，简便，高效的冷冻程序人胚泡

和无血清收集无饲养小鼠胚胎干细胞条件培养基的无细胞方法

Solea Senegalensis精子中活性氧的细胞内定位评价

对于斑马鱼精子超低温冷冻保存的一个高通量的方法和施肥

精子冷冻保存和恢复使用I ·冷冻包

全睾丸针浸泡超快速冷却法超低温保存斑马鱼细胞

使用软件和冷却装置进行鱼类精子评估

用于珊瑚精子冷冻保存以支持生物样本库和水产养殖的半自动化工作流程

宽吻海豚 (海豚截形) 精子: 收集、冷冻保存和外源体外受精