Veterinary School, Federal University of Goiás State, Goiânia, cp 131, Campus II, UFG, cep 74001-970, Brazil.
Anim Reprod Sci. 2010 Apr;118(2-4):176-81. doi: 10.1016/j.anireprosci.2009.08.015. Epub 2009 Sep 4.
The objectives of this study were to compare two different methods of evaluating bull sperm morphology, bright-field (BF) microscopy of eosin-nigrosin (EN) stained dry-mount semen smears and differential interference phase contrast (DIC) microscopy of wet-mount semen 'fixed' in isotonic formal saline, both at 1000x. Ejaculates (n=72) were evaluated, representing both pre- and post-breeding season ejaculates collected from 40 2-yr-old beef bulls via electro-ejaculation. For both methods, 200 sperm were counted in random fields with defects categorized as major (MAD) and minor (MID). Sperm abnormalities were also placed into two other categories: those considered to be most influenced by process (wet or dry, METHDEF) and those with depictions that could be influenced by optics (BF or DIC, OPTIDEF). Differences (P<0.05) occurred between DIC and BF methods respectively: MAD 23.3/16.1, MID 7.6/13.4, acrosome 3.8/1.1, midpiece 9.2/11.7, tail 2.0/4.7, droplets 8.3/4.2, METHDEF 14.2/21.4 and OPTIDEF 13.0/5.5, but not (P>0.05) in percent normal sperm 69.1/70.4 or sperm head defects 7.5/8.3. Acrosome, tail and droplet defects were observed in 98.2/80.5, 86.1/100 and 98.2/94.4 percent of bulls for DIC and BF, respectively (P<0.05). As percent normal sperm did not differ between methods, bright-field microscopy assessment of EN preparations was considered to be a satisfactory method to categorize breeding soundness of bulls. However, DIC was more effective in visualizing major defects, while BF (which included stained smear preparation) was considered to cause more minor defects. Thus DIC was considered to be the preferred method of semen assessment for accurate assessment of sperm morphology in bulls.
本研究的目的是比较两种不同的评估公牛精子形态的方法，一种是使用曙红-黑染（EN）干片的明场（BF）显微镜，另一种是使用在等渗福尔马林液中“固定”的湿片的相差干涉相衬（DIC）显微镜，均在 1000x 下观察。评估了 72 个精液样本，这些样本来自 40 头 2 岁的肉牛公牛，通过电刺激采集，分别来自繁殖前和繁殖后季节。对于这两种方法，在随机视野中分别计数 200 个精子，将缺陷分为主要缺陷（MAD）和次要缺陷（MID）。精子异常也分为两类：一类是认为最受处理（干湿，METHDEF）影响的，另一类是可以受到光学影响（BF 或 DIC，OPTIDEF）的。DIC 和 BF 方法之间存在差异（P<0.05）：MAD 分别为 23.3/16.1、MID 分别为 7.6/13.4、顶体分别为 3.8/1.1、中段分别为 9.2/11.7、尾部分别为 2.0/4.7、液滴分别为 8.3/4.2、METHDEF 分别为 14.2/21.4 和 OPTIDEF 分别为 13.0/5.5，但正常精子百分比 69.1/70.4 或精子头缺陷 7.5/8.3 没有差异（P>0.05）。98.2/80.5、86.1/100 和 98.2/94.4%的公牛在 DIC 和 BF 下分别观察到顶体、尾部和液滴缺陷（P<0.05）。由于两种方法的正常精子百分比没有差异，因此认为 EN 染色干片的 BF 显微镜评估是一种评估公牛繁殖力的满意方法。然而，DIC 更有效地观察到主要缺陷，而 BF（包括染色涂片制备）则被认为会导致更多的次要缺陷。因此，DIC 被认为是评估公牛精子形态的首选方法，可更准确地评估精子形态。
