Sperm preparation through Sephadex™ filtration improves in vitro fertilization rate of buffalo oocytes.
Sperm preparation through Sephadex™ filtration improves in vitro fertilization rate of buffalo oocytes.
复制标题
通过 Sephadex™ 过滤制备精子可提高水牛卵母细胞的体外受精率。
DOI：
10.1111/rda.13117
复制
发表时间：
2018
期刊：
影响因子：
--
通讯作者：
Husna AU
中科院分区：
文献类型：
--
作者：
Husna AU
通常，游动向上法用于分离高质量精子；然而，在离心过程中，长时间的处理和细胞间的密切接触是精子氧化应激的不可避免的因素。目的是评估Sephadex™和玻璃棉过滤分离水牛体外受精的活动、完整和活精子的效果。采用抽吸法从屠宰水牛卵巢中收集卵母细胞复合物（COCs），在38.5℃、5% CO2的CO2培养箱中成熟24小时。成熟的COCs在受精TALP中冲洗两次，并置于预热的无精子的受精培养基中。冷冻保存的水牛精液在37°C下解冻30 s，并通过Sephadex™、玻璃棉过滤和swim - up（对照）进行处理。评估总精子和活动精子恢复率，在受精TALP中重悬，在co2培养箱中孵育15-20分钟。每种方法制备的样品被分成两组：一组用于研究精子质量（渐进式活力、膜完整性、活力、可居性），另一组与10-15组体外成熟卵母细胞共孵育。精子质量数据采用方差分析，体外受精率采用卡方检验，使用SPSS‐20进行比较。采用最小显著性差异（LSD）检验比较治疗方法。玻璃棉过滤获得了更高的总精子和活动精子回收率，而Sephadex™过滤改善了精子质量（渐进式运动、膜完整性、活力和宜居性）（p< 0.05）。与玻璃棉过滤和游上（对照组）相比，通过Sephadex过滤的精子制备在卵裂率方面获得了更高的体外受精率。总之，通过Sephadex过滤选择的冷冻保存的Nili - Ravi水牛精子质量得到改善，体外成熟/受精卵母细胞的受精率（卵裂率）更高。凝胶过滤技术在水牛体外受精中应用前景广阔。
ContentsRoutinely, swim‐up method is used to separate high‐quality sperm; however, long processing time and close cell‐to‐cell contact during the centrifugation step are inevitable elements of oxidative stress to sperm. The objective was to evaluate Sephadex™and glass wool filtration to separate motile, intact and viable sperm for in vitro fertilization in buffalo. The cumulus–oocyte complexes (COCs) were collected from ovaries of slaughtered buffaloes by aspiration and matured for 24 hr in CO2incubator at 38.5°C and 5% CO2. Matured COCs were rinsed twice in fertilization TALP and placed in the pre‐warmed fertilization medium without sperm. Cryopreserved buffalo semen was thawed at 37°C for 30 s and processed through Sephadex™, glass wool filtration and swim‐up (control). Total and motile sperm recovery rates were assessed, resuspended in fertilization TALP and incubated for 15–20 min in CO2incubator. Samples prepared by each method were divided into two aliquots: one aliquot was studied for sperm quality (progressive motility, membrane integrity, viability, liveability), while the other was subjected to co‐incubation with sets of 10–15 in vitro matured oocytes. Data on sperm quality were analysed by ANOVA, while in vitro fertilizing rates were compared by chi‐squared test using SPSS‐20. Least significant difference (LSD) test was used to compare treatment means. Glass wool filtration yielded higher total and motile sperm recovery rate, while Sephadex™filtration improved (p<.05) sperm quality (progressive motility, membrane integrity, viability, liveability). Sperm preparation through Sephadex filtration yielded higher in vitro fertilization rate in terms of cleavage rate compared to glass wool filtration and swim‐up (control). In conclusion, cryopreserved Nili‐Ravi buffalo sperm selected through Sephadex filtration showed improved quality and yielded better fertilization rates (cleavage rate) of in vitro matured/fertilized oocytes. Sephadex filtration could be a promising technique for use in in vitro fertilization in buffalo.