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缘关系的风险。一种解决方案是精子冷冻保存。一些作者已经与冻存海豚精子5，，78，9，非常令人鼓舞的结果。此外，在 2005 年，第一次的设想通过人工受精方式利用冷冻的精子的小海豚诞生报告的10。几年后，这样做是使用性排序精子11。
海豚的精子有经低温保存使用不同的技术，从复杂的方法，如可编程冰箱5，常用的方法，如干冰12或液态氮蒸气2，9， 13。使用干冰或液氮蒸气的主要优点是设备的冷冻保存的精子提取，无需使用高度专业化的同一位置的可能性。精子冷冻在稻草上聚苯乙烯在液态氮蒸气中是简单，快速，和便宜的方法13。在实践中，当这种方法被用在了海豚，精子活率为 65%。然而，它是重要的是考虑建立一种规范的方法是多么的困难: 技术可能受到外部因素，如温度、 湿度、 聚苯乙烯大小等。因此，研究有必要进一步优化和规范程序，以提高使用的精子冷冻用液氮气相方法的成功。
为了优化冻结协议，它是重要发展精子功能评估的可靠方法。体外受精是测试精子受精的潜力的良好手段。理想情况下，海豚卵母细胞应该用于体外受精，但困难和艰辛的过程，需要从女性获得卵母细胞代表重要的约束。因此，利用外源体外受精的可能性打开一种新的手段来测试施肥效率在海豚。从其他物种的卵母细胞可以用于外源海豚体外受精。结果表明，外源体外受精牛卵母细胞和精子海豚之间可以进行。冻结和解冻的海豚精子可以穿透透明完整牛卵母细胞并生成一个混合胚胎。
用于收集协议提出相结合，冻融，表演牛卵母细胞与异体受精已被成功证明和代表冷冻保存的优化和评价的第一步海豚的精子。它是重要的是注意在这些程序中仍然有许多未知因素。然而，受赠海豚精子的能力使它可访问，并允许它被关在实验室。这将促进科学研究和可以导致更好地了解生理、 构成和精子的行为。
评价的外源体外受精的精子可以缓解的优化冻结协议，但它可能也用于将来测试和选择男性生育效率。一个重要的考虑因素是真正施肥数字和异体受精值之间的关系。此外，而新奇的外源体外受精，这两个系统遥远物种、 牛和海豚，之间可以建立新生物的假说。总之，这些协议开辟新的空间繁殖研究海豚和其他海洋哺乳动物。

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>FERT-TALP medium</td>
			<td>Merck</td>
			<td></td>
		</tr>
		<tr>
			<td>TCM-199</td>
			<td>Sigma</td>
			<td>M-4530</td>
		</tr>
		<tr>
			<td>Hoechst 33342</td>
			<td>Sigma</td>
			<td>B-2261</td>
		</tr>
		<tr>
			<td>4-well dishes</td>
			<td>Nunc</td>
			<td>176740</td>
		</tr>
		<tr>
			<td>Density gradient BoviPure</td>
			<td>Nidacon International</td>
			<td>BP-100</td>
		</tr>
		<tr>
			<td>Washing solution Boviwash</td>
			<td>Nidacon International</td>
			<td>BW-100</td>
		</tr>
		<tr>
			<td>Magnesium chloride</td>
			<td>Promega</td>
			<td>A35 1H</td>
		</tr>
		<tr>
			<td>Sterile water</td>
			<td>Mili Q sintesis A10 Millipore</td>
			<td>A35 1H</td>
		</tr>
		<tr>
			<td>Buffer Tris Borate EDTA</td>
			<td>Sigma</td>
			<td>T4415</td>
		</tr>
		<tr>
			<td>MB agarose</td>
			<td>Biotools</td>
			<td>20.012</td>
		</tr>
		<tr>
			<td>5X GoTaq flexi buffer</td>
			<td>Mili Q sintesis A10 Millipore</td>
			<td>M 890 A</td>
		</tr>
		<tr>
			<td>MB agarose</td>
			<td>Biotools</td>
			<td>20.012</td>
		</tr>
		<tr>
			<td>Taq polymerase</td>
			<td>Promega</td>
			<td></td>
		</tr>
		<tr>
			<td>SafeView</td>
			<td>NBS Biologicals Ltd.</td>
			<td>M 890 A</td>
		</tr>
		<tr>
			<td>Makler counting chamber</td>
			<td>Sefi Medical</td>
			<td></td>
		</tr>
		<tr>
			<td>Thoma chamber</td>
			<td>Hecht-Assistant</td>
			<td></td>
		</tr>
		<tr>
			<td>pHmeter MicropH 2000</td>
			<td>Crison Instruments</td>
			<td></td>
		</tr>
		<tr>
			<td>Osmometer Advanced micro osmometer 3300</td>
			<td>Norwood</td>
			<td></td>
		</tr>
		<tr>
			<td>Computer assisted sperm analysis system</td>
			<td>Projectes y Serveis R+D</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereomicroscope MZ 95</td>
			<td>Leica</td>
			<td></td>
		</tr>
		<tr>
			<td>Epifluorescent optics Eclipse Te300</td>
			<td>Nikon</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal assistant 4.02 software</td>
			<td>Bio-Rad</td>
			<td>3D analysis software</td>
		</tr>
		<tr>
			<td>Confocal laser scanning microscopy</td>
			<td>Bio-Rad</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropippetes (P2. P20, P200, P1000)</td>
			<td>Gilson</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge tubes</td>
			<td>VWR</td>
			<td></td>
		</tr>
		<tr>
			<td>UV iluminator</td>
			<td>Bio-Rad</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR Thermal cycler Primus 96 Plus</td>
			<td>MWG AG Biotech</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。
海豚精子有能力穿透去透明带完整牛卵母细胞和生产杂交胚胎
图 1A和表 1显示海豚附加到牛 ZP 2.5 h 的共同孵化后的精子。海豚精子被附着在百分比高于牛精子 （图 1B和表 1）。事实上，荧光显微镜观察表明，海豚精子能够穿透去透明带完整牛精子 (图 1)，导致原核形成和裂解 （图 1E和表 1）。共焦显微镜 （图 1和图 1 楼） 证实了这一点。没有多精受精前后从外源 IVF 组卵母细胞共同培养 (表 1) 12 小时。原核形成异源体外受精中的达到最高的百分比在 24h，同源组中，在共同培养 (表 1) 18 小时发生时间较长。在 48 小时后孵化的卵裂率是低于异种异体受精 (表 1)。自发的孤雌激活率 8.0%，观察牛成熟未受精卵卵母细胞在 48 h (表 1)。
最后，PCR 结果证实物质存在的海豚遗传杂交胚胎 (图 3) 通过评估编码核糖体 18 S 海豚参考基因的存在蛋白质 (图 2)。
<img alt="Figure 1" src="/files/ftp_upload/55237/55237fig1.jpg"> 
图 1: 海豚精子附着牛卵透明带 (ZP)。附加的海豚 (A) 和牛 (B) 精子与卵完整牛卵母细胞共同孵育的 2.5 小时后。配子是沾满赫斯特和可视化相衬显微镜 （放大 40 倍） 下。 Periviteline 牛卵母细胞 （C和D） 和 (E) 牛卵母细胞胞质中的 decondensing 海豚精子头部空间中的海豚精子。图像被捕获在共焦显微镜下，对应于赤道平面的推定的合子 （Hoechst 染色; 40 X 放大）。二极体 (F) 在相差显微镜下观察后外源体外受精技术组成的海豚精子与卵完整牛卵母细胞 （Hoechst 染色; 40 X 放大; 共同孵化 24 小时条码秤 = 25 微米)。图转载自参考文献3的权限。<a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55237/55237fig1large.jpg" target="_blank">请点击这里查看此图的大版本。</a>
<img alt="Figure 2" src="/files/ftp_upload/55237/55237fig2.jpg"> 
图 2: 代表电泳 （2%琼脂糖凝胶溴化乙锭染色） 显示 18 S 海豚基因的 PCR 扩增结果。车道 1 到 30 显示推定海豚/牛杂交胚胎;S1 和 S2 的车道功能 4 吴/mL 和 0.4 吴/mL 海豚精子 DNA，分别;和小路 C1 到 C5 功能两个细胞牛胚胎和 H2O （水） （空白对照）。箭头指示 190 bp 频段对应放大 18 S rDNA。图转载自参考文献3的权限。<a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55237/55237fig2large.jpg" target="_blank">请点击这里查看此图的大版本。</a>
<img alt="Figure 3" src="/files/ftp_upload/55237/55237fig3.jpg"> 
图 3: 代表图像的杂交胚胎。两个细胞杂交胚胎 (A) 在培养 （Hoechst 染色; 40 X 放大） 48 小时的形象代表。在 48 小时的潜伏期，在不同阶段的划分，在 (B) 体视显微镜下可视化污泥而不染的杂交胚胎。<a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55237/55237fig3large.jpg" target="_blank">请点击这里查看此图的大版本。</a>
<img alt="Table 1" src="/files/ftp_upload/55237/55237table1.jpg"> 
表 1: 附件、 多精受精、 原核形成和分别后与牛卵母细胞和牛或海豚的精子，同源及异源共同孵育的卵裂率。变量，采用方差分析 （秩和曼-惠特尼测试）。值表示为十二复制; 平均 ± SEM （范围）n = 卵母细胞或推定的受精卵检查的总数。表被转载从参考文献3的权限。<a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55237/55237table1large.jpg" target="_blank">请点击这里查看此图的大版本。</a>

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

Nanyang Technological University

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 ...

Isolation of Mouse Endometrial Epithelial and Stromal Cells for In Vitro Decidualization

Decidualization is a progesterone-dependent differentiation process of endometrial stromal cells and is a prerequisite for successful embryo implantation. Although many efforts have been made to reveal the underlying mechanisms of decidualization, the exact signaling between the epithelial cells that are in contact with the embryo and the underlying stromal cells remains poorly understood. Therefore, studying decidualization in a way that takes both the epithelial and stromal cells into account could improve our knowledge about the molecular details of decidualization. For this purpose, in vivo models of artificial decidualization are physiologically the most relevant; however, manipulation of intercellular communication is limited. Currently, in vitro cultures of endometrial stromal cells are being used to investigate the modulation of decidualization by several signaling molecules. Conventionally, human or mouse endometrial stromal cells are used. However, the availability of human samples is very often limited. Furthermore, the use of murine tissues is accompanied with variety in the method of culturing. This study presents a validated and standardized method to obtain pure Endometrial Epithelial Cell (EEC) and Stromal Cell (ESC) cultures using adult intact mice treated with estrogen for three consecutive days. The protocol is optimized to improve the yield, viability, and purity of the cells and was further extended in order to study decidualization in a coculture of EEC and ESC. This model may be suitable to exploit the importance of both cell types in decidualization and to evaluate the contribution of significant signaling molecules secreted by EEC or ESC during the intercellular communication.

Isolation of Mouse Endometrial Epithelial and Stromal Cells for In Vitro Decidualization

This study presents a standardized and validated method for the isolation and culture of primary mouse endometrial stromal and epithelial cells, which can be used in a coculture system to study in vitro decidualization.

鼠标子宫内膜上皮细胞和基质细胞的隔离<em&gt;体外</em&gt;蜕膜

Decidualization is a progesterone-dependent differentiation process of endometrial stromal cells and is a prerequisite for successful embryo implantation. ...

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) ...

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the proteins involved in these pathways are evolutionarily conserved between mammals and other eukaryotes, such as the fruit fly Drosophila melanogaster, suggesting that similar organizing principles exist during the development of these organisms. Importantly, Drosophila has been used extensively to identify cellular and molecular mechanisms regulating processes that are required in mammals including neurogenesis, differentiation, axonal guidance, and synaptogenesis. Flies have also been used successfully to model a variety of human neurodevelopmental diseases. Here we describe a protocol for the step-by-step microdissection, fixation, and immunofluorescent localization of proteins within the adult Drosophila brain. This protocol focuses on two example neuronal populations, mushroom body neurons and retinal photoreceptors, and includes optional steps to trace individual mushroom body neurons using Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. Example data from both wild-type and mutant brains are shown along with a brief description of a scoring criteria for axonal guidance defects. While this protocol highlights two well-established antibodies for investigating the morphology of mushroom body and photoreceptor neurons, other Drosophila brain regions and the localization of proteins within other brain regions can also be investigated using this protocol.

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

This protocol describes the dissection and immunostaining of adult Drosophila melanogaster brain tissues. Specifically, this protocol highlights the use of Drosophila mushroom body and photoreceptor neurons as example neuronal subsets that can be accurately used to uncover general principles underlying many aspects of neuronal development.

成人果蝇脑内蘑菇体和感光神经元的解剖和荧光染色

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the ...

Collection of Post-mating Semen from the Female Reproductive Tract and Measurement of Semen Liquefaction in Mice

In mice, ejaculated semen is deposited in the uterus. After ejaculation, the semen changes consistency from gel-like to watery, a process called liquefaction. In this study, we show how to collect the post-ejaculated semen from the female reproductive tract in a mouse model. First, adult female mice in the estrus stage were housed in a male&#39;s cage overnight. The next morning, copulation was confirmed by the presence of copulatory plug at the vaginal opening. Female mice with copulatory plugs were euthanized, and each reproductive tract was collected as a whole (vagina, uterus, oviducts, ovaries), ensuring a closed system to contain the semen. The reproductive tract was placed in a 1.5 mL microcentrifuge tube, and the vagina was cut off to release the semen into the tube. To ensure maximum semen volume for analysis, toothless forceps were used to squeeze the uterine horns from ovarian end to vaginal end expelling remaining semen. The whole reproductive tract was then discarded. The semen-containing tube was briefly spun down. A 25 &#956;L capillary pipette was placed into the tube at a 180&#176; angle (parallel to the tube wall). The amount of time used to fill the capillary tube to the 25 &#956;L line was recorded. Semen from a proven male breeder usually takes approximately 60-180 s to fill a 25 &#956;L capillary tube. This semen collection technique can also be used in other downstream applications such as sperm imaging and motility analysis.

Semen liquefaction is required for sperm to be liberated from the seminal gel. This study provides the procedures for collecting semen from the female reproductive tract post-coitus, and measuring semen liquefaction time.

雌性生殖道交配后精子的收集及对小鼠精子液化的测定

In mice, ejaculated semen is deposited in the uterus. After ejaculation, the semen changes consistency from gel-like to watery, a process called ...

Handling and Treatment of Male European Eels (Anguilla anguilla) for Hormonal Maturation and Sperm Cryopreservation

During the last years, several research groups have been working on the development and improvement of new protocols for the European eel handling and maturation. As of yet, weekly injections of human chorionic gonadotropin (hCG) have proved to maturate males after just 5-6 weeks of treatment, producing high volumes of high-quality sperm during several weeks. In addition, sperm cryopreservation protocols using different extenders, cryoprotectants and cooling and thawing times have been previously described for European eel. Here, we show that Tanaka&#180;s extender solution can be directly used for fertilization or for cryopreservation, making unnecessary the usage of different types of solutions and dilutions. Furthermore, the use of methanol as a cryoprotectant makes this protocol easy to use as methanol has low toxicity and does not activate the sperm. The sperm does not need to be cryopreserved immediately after the addition of the cryoprotectant, and it can be used long after being thawed. Moreover, sperm motility is still high after thawing although it is lower than that of fresh sperm. The aim of this work is to show the best available protocol for European eel handling, maturation, and sperm cryopreservation.

Handling and Treatment of Male European Eels Anguilla anguilla for Hormonal Maturation and Sperm Cryopreservation

Protocols for European eel maturation and sperm cryopreservation have been improved over the last years. This article describes the best protocol available using human chorionic gonadotropin (hCG) for inducing maturation and methanol as cryoprotectant.

对雄性欧洲鳗鲡 (安圭拉) 进行激素成熟和精子冷冻保存的处理和处理

During the last years, several research groups have been working on the development and improvement of new protocols for the European eel handling and ...

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell types include enzyme-secreting acinar cells, an arborized ductal system responsible for the transportation of enzymes to the gut, and hormone-producing endocrine cells. 
Endocrine beta-cells are the sole cell type in the body that produce insulin to lower blood glucose levels. Diabetes, a disease characterized by a loss or the dysfunction of beta-cells, is reaching epidemic proportions. Thus, it is essential to establish protocols to investigate beta-cell development that can be used for screening purposes to derive the drug and cell-based therapeutics. While the experimental investigation of mouse development is essential, in vivo studies are laborious and time-consuming. Cultured cells provide a more convenient platform for screening; however, they are unable to maintain the cellular diversity, architectural organization, and cellular interactions found in vivo. Thus, it is essential to develop new tools to investigate pancreatic organogenesis and physiology.
Pancreatic epithelial cells develop in the close association with mesenchyme from the onset of organogenesis as cells organize and differentiate into the complex, physiologically competent adult organ. The pancreatic mesenchyme provides important signals for the endocrine development, many of which are not well understood yet, thus difficult to recapitulate during the in vitro culture. Here, we describe a protocol to culture three-dimensional, cellular complex mouse organoids that retain mesenchyme, termed pancreatoids. The e10.5 murine pancreatic bud is dissected, dissociated, and cultured in a scaffold-free environment. These floating cells self-assemble with mesenchyme enveloping the developing pancreatoid and a robust number of endocrine beta-cells developing along with the acinar and the duct cells. This system can be used to study the cell fate determination, structural organization, and morphogenesis, cell-cell interactions during organogenesis, or for the drug, small molecule, or genetic screening.

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

Here, we present a protocol to generate insulin expressing 3D murine pancreatoids from free-floating e10.5 dissociated pancreatic progenitors and the associated mesenchyme.

无支架三维胰岛素表达 Pancreatoids 的实验研究体外培养的小鼠胰祖细胞

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell ...

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

鼠标子宫内膜上皮细胞和基质细胞的隔离

Solea Senegalensis精子中活性氧的细胞内定位评价

成人果蝇脑内蘑菇体和感光神经元的解剖和荧光染色

雌性生殖道交配后精子的收集及对小鼠精子液化的测定

对雄性欧洲鳗鲡 (安圭拉) 进行激素成熟和精子冷冻保存的处理和处理

无支架三维胰岛素表达 Pancreatoids 的实验研究体外培养的小鼠胰祖细胞

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

鼠标子宫内膜上皮细胞和基质细胞的隔离

Solea Senegalensis精子中活性氧的细胞内定位评价

成人果蝇脑内蘑菇体和感光神经元的解剖和荧光染色

雌性生殖道交配后精子的收集及对小鼠精子液化的测定

对雄性欧洲鳗鲡 (安圭拉) 进行激素成熟和精子冷冻保存的处理和处理

无支架三维胰岛素表达 Pancreatoids 的实验研究体外培养的小鼠胰祖细胞

宽吻海豚 (海豚截形) 精子: 收集、冷冻保存和外源体外受精