冷冻保护剂和复苏方案对用于卵胞浆内单精子注射剂量的再次冷冻的冻融种马精子活力的影响。
Effects of extender, cryoprotectants and thawing protocol on motility of frozen-thawed stallion sperm that were refrozen for intracytoplasmic sperm injection doses.
机构信息
Department of Biomedical Sciences, Colorado State University, Equine Reproduction Laboratory, 3101 Rampart Road, Fort Collins, CO, 80521, USA.
Graduate Program in Animal Medicine (Equine), Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil.
出版信息
Theriogenology. 2019 Sep 15;136:36-42. doi: 10.1016/j.theriogenology.2019.06.030. Epub 2019 Jun 19.
We examined the effects of different freezing extenders, cryoprotectant agents (CPA) and initial thawing temperatures for preparing doses of refrozen stallion sperm for intracytoplasmic sperm injection (ICSI). Single ejaculates, from twelve stallions, were frozen in lactose-EDTA-egg yolk extender (LE) with 5% glycerol. In experiment 1, sperm were initially thawed to 5 °C or 37 °C, before being diluted in LE or skim milk-egg yolk extender (SMEY) containing either 5% glycerol (GLY), 5% methylformamide (MF) or 5% of a combination of both (GMF). In experiment 2, frozen sperm were initially thawed to 5 °C, diluted and refrozen in SMEY containing 2, 4, 6 or 8% GLY or GMF. In Experiment 1, sperm motility was reduced after each cryopreservation cycle (P < 0.05). Extender type did not affect motility after refreezing (P > 0.05), but sperm initially thawed to 5 °C exhibited higher motility than sperm thawed to 37 °C (P < 0.05). In addition, sperm refrozen in SMEY containing MF or GMF exhibited higher motility than sperm refrozen in GLY alone (P < 0.05). In experiment 2, there was an interaction between CPA and CPA concentration (P < 0.05). Sperm refrozen with GMF had higher motility than refrozen sperm with GLY (P < 0.05), and while GLY concentration did not affect post-thaw motility (P > 0.05). Sperm refrozen with 6 or 8% GMF exhibited the highest motility (P < 0.05). In conclusion, sperm motility is best maintained when thawing and refreezing stallion sperm in low sperm concentration ICSI doses by initially thawing the sperm to 5 °C and diluting the sperm in a freezing extender with 8% GMF.
我们研究了不同的冷冻液、冷冻保护剂（CPA）和初始解冻温度对制备用于胞浆内精子注射（ICSI）的重冷冻种马精子剂量的影响。从 12 头种马采集的单个精子用乳糖-EDTA-卵黄液（LE）加 5%甘油冷冻。在实验 1 中，精子最初在 5°C 或 37°C 解冻，然后在含有 5%甘油（GLY）、5%甲酰胺（MF）或两者的组合（GMF）的 LE 或脱脂奶-卵黄液（SMEY）中稀释。在实验 2 中，冷冻精子最初在 5°C 解冻，然后在含有 2%、4%、6%或 8% GLY 或 GMF 的 SMEY 中稀释和重冷冻。在实验 1 中，每次冷冻保存周期后精子活力都会降低（P<0.05）。重冷冻后，不同的冷冻液类型对精子活力没有影响（P>0.05），但在 5°C 解冻的精子活力高于在 37°C 解冻的精子活力（P<0.05）。此外，在含有 MF 或 GMF 的 SMEY 中重冷冻的精子活力高于单独在 GLY 中重冷冻的精子活力（P<0.05）。在实验 2 中，CPA 和 CPA 浓度之间存在相互作用（P<0.05）。与 GLY 相比，用 GMF 重冷冻的精子活力更高（P<0.05），而 GLY 浓度对解冻后精子活力没有影响（P>0.05）。用 6%或 8% GMF 重冷冻的精子活力最高（P<0.05）。结论：当在低浓度的 ICSI 剂量中解冻和重冷冻种马精子时，最初将精子在 5°C 解冻，并在含有 8% GMF 的冷冻液中稀释精子，这样可以更好地保持精子活力。
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