Gliozzi T M, Luzi F, Cerolini S
Istituto di Biologia e Biotecnologia Agraria-CNR, via Bassini, 15-20133 Milano, Italy.
Theriogenology. 2003 Sep 1;60(4):635-45. doi: 10.1016/s0093-691x(03)00082-7.
The ethidium bromide (EtBr) exclusion procedure, a fluorometric method for measuring sperm cell viability, was studied to optimize the use of this technique on boar, rabbit and rooster semen. Diluted semen was used for boars and roosters. Diluted rabbit semen did not allow for reliable fluorescence readings; the interference of granules characteristic of rabbit seminal plasma was suggested as its cause. Therefore, rabbit semen was washed on several Percoll and Optiprep density gradients, with the aim of removing the granules from the sperm suspension. The complete absence of granules was not obtained, however, the best result was provided by the 35/70% Percoll density gradient. Most spermatozoa formed a loose pellet with low contamination. Although the washing procedure resulted in a selective action, Percoll washed semen was used to assess the EtBr procedure. The fluorescence intensities of stained fresh and stained digitonin-permeabilized samples were corrected, respectively, for the nonspecific fluorescence measures of fresh and digitonin-permeabilized samples both unstained. The contribution of the dye was subtracted from the corrected values, then the ratio between the corrected values of fresh and permeabilized cells provided the proportion of damaged cells in the sample. The working cell concentration range giving a constant proportion of damaged cells was set using diluted semen for boars and roosters (8-32 x 10(6) cell/ml) and Percoll washed semen for rabbits (4-16 x 10(6) cell/ml). The reliability of the fluorometric method was compared with the traditional nigrosin-eosin (NE) staining technique. The intactness of sperm samples containing known proportions of fresh and killed cells was measured in defined working cell ranges. For boars and roosters the values determined by fluorometry agreed closely with those determined using the NE method.
研究了溴化乙锭（EtBr）排除法（一种用于测量精子细胞活力的荧光法），以优化该技术在公猪、兔子和公鸡精液上的应用。公猪和公鸡使用稀释精液。稀释的兔精液无法获得可靠的荧光读数；兔精浆特有的颗粒干扰被认为是其原因。因此，将兔精液在几个Percoll和Optiprep密度梯度上进行洗涤，目的是从精子悬液中去除颗粒。然而，并未完全去除颗粒，不过35/70%的Percoll密度梯度提供了最佳结果。大多数精子形成了污染低的松散沉淀。尽管洗涤过程具有选择性作用，但仍使用Percoll洗涤后的精液来评估EtBr法。分别针对未染色的新鲜样本和洋地黄皂苷通透处理样本的非特异性荧光测量值，对染色的新鲜样本和染色的洋地黄皂苷通透处理样本的荧光强度进行校正。从校正值中减去染料的贡献，然后新鲜细胞和通透处理细胞校正值之间的比率给出了样本中受损细胞的比例。使用公猪和公鸡的稀释精液（8 - 32×10⁶个细胞/毫升）以及兔子的Percoll洗涤精液（4 - 16×10⁶个细胞/毫升）设定能给出恒定受损细胞比例的工作细胞浓度范围。将荧光法的可靠性与传统的黑色素 - 伊红（NE）染色技术进行比较。在确定的工作细胞范围内测量含有已知比例新鲜细胞和死亡细胞的精子样本的完整性。对于公猪和公鸡，荧光法测定的值与使用NE法测定的值非常接近。
