The effects of cooling rates and type of freezing extenders on cryosurvival of rat sperm.
The effects of cooling rates and type of freezing extenders on cryosurvival of rat sperm.
复制标题
冷却速率和冰点延长剂类型对大鼠精子的冷冻保护物的影响。
DOI：
10.1016/j.cryobiol.2013.05.009
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发表时间：
2013-10
期刊：
影响因子：
2.7
通讯作者：
Agca, Yuksel
中科院分区：
文献类型：
--
作者：
Varisli, Omer;Scott, Hollie;Agca, Cansu;Agca, Yuksel
由于大鼠精子对各种应激因素敏感，其冷冻保存非常具有挑战性。本研究的目的是确定远交 Sprague Dawley (SD) 和近交 Fischer 344 (F344) 大鼠品系附睾精子的最佳冷却速率和延长剂。将 10-12 周龄性成熟 SD 和 F344 菌株的附睾精子悬浮在五种不同的冷冻稀释剂中，即 HEPES 缓冲的 Tyrode 乳酸 (TL-HEPES)、改良 Kreb’s Ringer 碳酸氢盐 (mKRB)、3% 脱水脱脂牛奶 (SM)、Salamon’s Tris-柠檬酸盐 (TRIS) 和 tes/tris (TES)。所有增量剂均含有 20% 蛋黄、0.75% Equex Paste 和 0.1 M 棉子糖或 0.1 M 蔗糖。将每个扩展器中的精子样本冷却至 4°C，并在冷冻前保持 45 分钟以达到平衡。将每个扩展器中平衡的精子样本放置在插入 Linkam Cryostage (BCS 196) 的浅石英盘上。然后使用不同的冷却速率（10、40、70 和 100 °C/min）将样品冷却至最终温度 -150 °C。为了解冻，将含有精子样本的石英盘从 Linkam 冷冻台中快速取出，并放置在 37°C 载玻片加热器上，并在运动分析前保持 1 分钟。分别通过 SYBR-14/碘化丙啶、Alexa Fluor-488-PNA 缀合物和 JC-1 评估精子膜和顶体完整性以及线粒体膜电位 (MMP)。比较冷却速率和延长剂之间的总运动性、顶体完整性、膜完整性和 MMP 值。冷却速率和稀释剂类型对冷冻存活有显着影响（P<0.05）。两种菌株的精子活力随着冷却速率的增加而增加（P<0.05）。当 100 °C/min 的冷却速率与含有 20% 蛋黄、0.75% Equex 糊剂和 0.1 M 蔗糖或棉子糖的 TES 增量剂结合使用时，可实现最高的冷冻存活率 (P < 0.05)。本研究表明，含有 0.1 M 棉子糖或蔗糖的 TES 稀释剂以 70 °C /min 和 100 °C /min 的冷却速率改善了大鼠精子解冻后的活力。
Cryopreservation of rat sperm is very challenging due to its sensitivity to various stress factors. The objective of this study was to determine the optimal cooling rate and extender for epididymal sperm of outbred Sprague Dawley (SD) and inbred Fischer 344 (F344) rat strains. The epididymal sperm from 10–12 weeks old sexually mature SD and F344 strains were suspended in five different freezing extenders, namely HEPES buffered Tyrode’s lactate (TL-HEPES), modified Kreb’s Ringer bicarbonate (mKRB), 3% dehydrated skim milk (SM), Salamon’s Tris-citrate (TRIS), and tes/tris (TES). All extenders contained 20% egg yolk, 0.75% Equex Paste and 0.1 M raffinose or 0.1 M sucrose. The sperm samples in each extender were cooled to 4°C and held for 45 min for equilibration before freezing. The equilibrated sperm samples in each extender were placed onto a shallow quartz dish inserted into Linkam Cryostage (BCS 196). The samples were then cooled to a final temperature of −150 °C by using various cooling rates (10, 40, 70, and 100 °C/min). For thawing, the quartz dish containing the sperm samples were rapidly removed from the Linkam cryo-stage and placed on a 37 °C slide warmer and held for 1 min before motility analysis. Sperm membrane and acrosomal integrity and mitochondrial membrane potential (MMP) were assessed by SYBR-14/Propidium iodide, Alexa Fluor-488-PNA conjugate and JC-1, respectively. The total motility, acrosomal integrity, membrane integrity and MMP values were compared among cooling rates and extenders. Both cooling rate and type of extender had significant effect on cryosurvival (P<0.05). Sperm motility increased as cooling rate was increased for both strains (P<0.05). Highest cryosurvival was achieved when 100 °C/min cooling rate was used in combination with TES extender containing 20% egg yolk, 0.75% Equex paste and either 0.1 M sucrose or raffinose (P < 0.05). This study showed that TES extender containing 0.1 M raffinose or sucrose with 70 °C /min and 100 °C /min cooling rate improved post-thaw motility of rat sperm.
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影响因子：
2.2
作者：
Graham, JK
通讯作者：
Graham, JK
影响因子：
6.1
作者：
Gunasena, KT;Villines, PM;Critser, JK
通讯作者：
Critser, JK
影响因子：
2.7
作者：
Kim, Suhee;Agca, Cansu;Agca, Yuksel
通讯作者：
Agca, Yuksel
影响因子：
1.5
作者：
Barbas, J. P.;Mascarenhas, R. D.
通讯作者：
Mascarenhas, R. D.
DOI：
10.1016/b978-012074903-4/50010-8
发表时间：
2006-01-01
期刊：
LABORATORY RAT, 2ND EDITION
影响因子：
--
作者：
Agca, Yuksel;Critser, John K.
通讯作者：
Critser, John K.