Filliers M, Rijsselaere T, Bossaert P, De Causmaecker V, Dewulf J, Pope C E, Van Soom A
Department of Reproduction, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium.
Theriogenology. 2008 Dec;70(9):1550-9. doi: 10.1016/j.theriogenology.2008.07.004. Epub 2008 Aug 8.
Epididymal cat sperm is commonly used for in vitro fertilization. Because of the high variability in preparation protocols and methods of evaluation, sperm quality may vary considerably between experiments and laboratories. The aims of the present study were (1) to describe an epididymal sperm preparation protocol to produce clean, highly motile samples using density gradient centrifugation, (2) to provide reference values of computer-assisted semen analysis (CASA) parameters of fresh epididymal cat sperm after density gradient centrifugation and (3) to investigate the effect of cool storage on various spermatozoa characteristics. After slicing the epididymides, viable and motile sperm cells were isolated using Percoll centrifugation. Sperm motility parameters were subsequently assessed using CASA in experiment 1. In experiment 2, fresh (day 0) sperm samples were evaluated for motility parameters (HTR) and stained for assessment of acrosomal status (FITC-PSA), morphology (eosin/nigrosin (E/N)), membrane integrity (E/N and SYBR((R))14-PI) and DNA fragmentation (TUNEL). After addition of a Tris-glucose-citrate diluent containing 20% egg yolk, samples were cooled to 4 degrees C and reassessed on d1, d3, d5, d7 and d10. Cool storage impaired most motility and velocity parameters: MOT, PMOT, VAP, VSL, VCL, BCF, RAPID and the percentage of normal spermatozoa showed a decrease over time (P<0.05) as compared to fresh samples. In contrast, STR, ALH, membrane integrity, DNA fragmentation and the percentage of acrosome intact spermatozoa were not affected by cool storage. However, the influence of cool storage of cat spermatozoa on subsequent in vitro embryo development and quality after IVF requires further investigation.
附睾猫精子常用于体外受精。由于制备方案和评估方法的高度变异性，不同实验和实验室之间的精子质量可能有很大差异。本研究的目的是：（1）描述一种使用密度梯度离心法制备清洁、高活力样本的附睾精子制备方案；（2）提供密度梯度离心后新鲜附睾猫精子的计算机辅助精液分析（CASA）参数的参考值；（3）研究冷藏对各种精子特征的影响。将附睾切片后，使用Percoll离心法分离活的和有活力的精子细胞。随后在实验1中使用CASA评估精子活力参数。在实验2中，对新鲜（第0天）精子样本进行活力参数（HTR）评估，并进行染色以评估顶体状态（FITC-PSA）、形态（伊红/美蓝（E/N））、膜完整性（E/N和SYBR®14-PI）和DNA片段化（TUNEL）。加入含有20%蛋黄的Tris-葡萄糖-柠檬酸盐稀释剂后，将样本冷却至4℃，并在第1天、第3天、第5天、第7天和第10天重新评估。冷藏损害了大多数活力和速度参数：MOT、PMOT、VAP、VSL、VCL、BCF、RAPID，与新鲜样本相比，正常精子百分比随时间下降（P<0.05）。相比之下，STR、ALH、膜完整性、DNA片段化和顶体完整精子百分比不受冷藏影响。然而，猫精子冷藏对后续体外胚胎发育和体外受精后胚胎质量的影响需要进一步研究。
