Liu Yue, Torres Leticia, Tiersch Terrence R
Aquatic Germplasm and Genetic Resources Center, School of Renewable Natural Resources, Louisiana State University Agricultural Center, Baton Rouge, LA, USA.
Aquatic Germplasm and Genetic Resources Center, School of Renewable Natural Resources, Louisiana State University Agricultural Center, Baton Rouge, LA, USA.
Theriogenology. 2018 Feb;107:50-56. doi: 10.1016/j.theriogenology.2017.10.037. Epub 2017 Oct 28.
Standardized evaluation of sperm quality is essential for research, commercial-scale cryopreservation, and induced spawning. However, standardized methods for evaluation of sperm bundles (spermatozeugmata or spermatophores) have not been established. The purpose of the present study was to use Redtail Splitfin (Xenotoca eiseni) as a model for freshwater livebearing fishes to establish initial standardized methods to collect sperm bundles, and quantitatively and qualitatively evaluate quality-related attributes. No sperm or sperm bundles were able to be collected by stripping. Testes were removed, rinsed, weighed, placed in 50 μL of buffer solution on a glass slide, and crushed gently 3-5 times with angled spade-tip forceps. Sperm bundles were released into the buffer solution and collected with a pipette into 1.5-mL centrifuge tubes. To quantify size and shape, images of bundles were captured with a CCD camera connected to a microscope, and measured with computer software. There was no significant correlation between body wet weight and major bundle axis length (P = 0.6759), minor axis length (P = 0.5658), average axis length (P = 0.5869), aspect ratio (P = 0.7839), and observed area (P = 0.5727). The concentrations of sperm bundles, estimated with the three methods (Makler counting chamber, a hemocytometer, and direct counting) were significantly different (P < 0.0001). Hemocytometers were suitable for estimation of bundles from X. eiseni. To evaluate activation of sperm, bundles were viewed with a microscope, and classified into one of five phases by evaluating morphology of the bundles and motion of sperm within the bundles as Phase 0 through Phase 4 that represented early through late activation stages. The frequencies and duration of each activation phase were used to evaluate dissociation of sperm bundles and motility capability of sperm within the bundles. Within 180 min of activation, all five phases were observed. Overall, this study for the first time established standardized methods to collect and evaluate quality-related attributes of sperm bundles. These standardized evaluations provide a basis for further modification, standardization, and generalization, which are useful in research on livebearing fishes involving male gametes, such as studies on cryopreservation, artificial insemination, and in development of germplasm repositories for imperiled species including goodeids.
精子质量的标准化评估对于研究、商业规模的冷冻保存和诱导产卵至关重要。然而，尚未建立评估精子束（精子团或精荚）的标准化方法。本研究的目的是以红尾裂鳍鱼（Xenotoca eiseni）作为淡水胎生鱼类的模型，建立收集精子束的初步标准化方法，并对与质量相关的属性进行定量和定性评估。通过挤压无法收集到精子或精子束。取出睾丸，冲洗、称重，置于载玻片上的50 μL缓冲溶液中，用带角度的铲形镊子轻轻挤压3 - 5次。精子束释放到缓冲溶液中，用移液器收集到1.5 mL离心管中。为了量化大小和形状，用连接到显微镜的CCD相机拍摄精子束的图像，并用计算机软件进行测量。鱼体湿重与精子束的长轴长度（P = 0.6759）、短轴长度（P = 0.5658）、平均轴长度（P = 0.5869）、纵横比（P = 0.7839）和观察面积（P = 0.5727）之间均无显著相关性。用三种方法（Makler计数室、血细胞计数器和直接计数）估算的精子束浓度存在显著差异（P < 0.0001）。血细胞计数器适用于估算红尾裂鳍鱼的精子束。为了评估精子的激活情况，用显微镜观察精子束，并根据精子束的形态和束内精子的运动情况将其分为五个阶段之一，即代表从早期到晚期激活阶段的0期到4期。每个激活阶段的频率和持续时间用于评估精子束的解离和束内精子的运动能力。在激活的180分钟内，观察到了所有五个阶段。总体而言，本研究首次建立了收集和评估精子束与质量相关属性的标准化方法。这些标准化评估为进一步的改进、标准化和推广提供了基础，有助于涉及雄配子的胎生鱼类研究，如冷冻保存、人工授精研究以及包括古氏鱼在内的濒危物种种质库的开发。
