Abstract：
This study aimed to select high-quality spermatozoa by sperm separation by magnetic activation of the fresh equine semen, compared to density gradient centrifugation and evaluating cell quality after selection. The semen of 10 stallions was collected by the artificial vagina technique. The samples analyzed were: (1) fresh semen; (2) density gradient centrifugation (DGC); (3) separation by magnetic activation (MASS) (nonapoptotic portion NAP); (4) separation by MASS (apoptotic portion-APT). Was analyzed: motility (light microscopy), concentration (Neubauer chamber), semen morphology (humid chamber in phase contrast), and supravital test (eosin/nigrosine). In DGC, 20 × 106 spermatozoa were used in the gradient of Percoll at 90% and 45% (400 μL each), centrifugation at 900 G/5 min, the pellet was diluted in HEPES. In MASS, 10 × 106 spermatozoa were diluted in 1.5 mL of HEPES, centrifugation at 300 G/10 min, pellet was resuspended in 150 μL of HEPES with 20 μL of nanoparticles bound to annexin V, incubation for 15 minutes and filtered in the magnetic separation column. The nonapoptotic fraction was collected directly and the apoptotic fraction after removal the column from the magnet and adding 300 μL of HEPES. The total abnormalities were 43.2% ± 2.78%, with the DGC and MASS being effective in reducing sperm abnormality by 15.6% ± 2.10% and 24.30% ± 1.63%, respectively, like the observed for the number of cells with intact membranes (50% lower in the APT portion). This nanotechnological method is efficient in producing high-quality semen samples for assisted reproduction procedures.
摘要：
本研究旨在通过新鲜马精液的磁激活精子分离，筛选出优质精子。与密度梯度离心法相比，并在选择后评估细胞质量。通过人工阴道技术收集了十匹种马的精液。分析的样品为:1)新鲜精液;2)密度梯度离心(DGC);3)通过磁活化分离(MASS)(非凋亡部分NAP);4)通过MASS分离(凋亡部分-APT)。分析：运动性（光学显微镜），浓度（Neubauer室），精液形态学（相位对比湿室），和超生命测试（伊红/苯胺黑）。在DGC中,20×106个精子用于90%和45%的Percoll梯度（每个400μl），900G/5分钟离心，将沉淀在HEPES中稀释。在大众中，将10×106个精子稀释在1.5mlHEPES中，300G/10分钟离心，将沉淀重悬于150μl的HEPES中，其中20μl的纳米颗粒与膜联蛋白V结合，孵育15分钟并在磁性分离柱中过滤。直接收集非凋亡部分,并在从磁体中取出柱并加入300μlHEPES后收集凋亡部分。总异常率为43.2±2.78%,DGC和MASS可有效减少15.6±2.10%和24.30±1.63%的精子异常，分别,与观察到的具有完整膜的细胞数量相似（APT部分降低50％）。这种纳米技术方法可以有效地生产用于辅助生殖程序的高质量精液样品。
