Department of Medicine and Animal Surgery, Faculty of Veterinary Science, University of Murcia, E-30100 Murcia, Spain.
Theriogenology. 2012 Sep 15;78(5):1117-25. doi: 10.1016/j.theriogenology.2012.05.008. Epub 2012 Jul 3.
The aim of this experimental study was to evaluate the effectiveness of sperm selection using single-layer centrifugation (SLC) prior to freezing on the sperm cryosurvival of boar ejaculates. Twenty-four sperm rich ejaculate fractions (SREF), collected from 24 boars (one per boar), were divided into two groups according to their initial semen traits: standard (n = 15) and substandard (n = 9). Semen samples from each SREF were split in two aliquots, one remained untreated (control samples) and the other was single-layer centrifuged (500 g for 20 min) using 15 mL of Androcoll-P Large (SLC samples). The yield of total, motile (assessed by CASA) and viable (cytometrically evaluated after staining with H-42, propidium iodide (PI) and FITC-PNA) sperm after SLC was higher (P < 0.05) in standard than substandard semen samples. The semen samples were cryopreserved using a standard 0.5-mL straw freezing protocol. Post-thaw sperm motility and viability (assessed at 30 and 150 min post-thawing) were higher (P < 0.05) in SLC than in control samples, regardless of the initial semen traits of the ejaculates. Additionally, thawed spermatozoa from SLC samples were more resistant (P < 0.05) to lipid peroxidation (BIOXYTECH MDA-586 Assay Kit) than those from control samples, regardless of the initial semen traits of the ejaculates. The SLC-treatment also influenced the functionality of thawed spermatozoa undergoing an in vitro capacitation process. The percentage of viable sperm showing high membrane fluidity (assessed with merocyanine 540) was lower (P < 0.05) in the SLC than in the control samples, regardless of the initial semen traits of the ejaculates. Thawed viable spermatozoa of SLC samples generated less (P < 0.05) reactive oxygen species (assessed with CM-H(2)DCFDA) than those of control samples in the substandard ejaculates. These findings indicate that the sperm selection before freezing using SLC improves the freezability of boar sperm.
本实验研究旨在评估在冷冻前使用单层离心（SLC）对猪精液精子冷冻存活率的影响。将 24 个猪精液富集部分（SREF）（每个猪 1 个）根据其初始精液特征分为两组：标准组（n=15）和次标准组（n=9）。每个 SREF 的精液样本被分成两份，一份未经处理（对照样本），另一份使用 15 mL 的 Androcoll-P Large 进行单层离心（500 g 离心 20 min）（SLC 样本）。SLC 后，标准精液样本的总精子、运动精子（通过 CASA 评估）和存活精子（用 H-42、碘化丙啶（PI）和 FITC-PNA 染色后通过细胞计量评估）的产量均高于次标准精液样本（P<0.05）。使用标准的 0.5-mL straw 冷冻方案冷冻精液样本。解冻后 30 和 150 分钟时，SLC 样本的精子活力和活力（评估）均高于对照组样本，与精液样本的初始精液特征无关。此外，SLC 样本解冻后的精子对脂质过氧化的抵抗力更强（P<0.05）（BIOXYTECH MDA-586 测定试剂盒），与精液样本的初始精液特征无关。SLC 处理还影响经历体外获能过程的解冻精子的功能。使用 mertocyanine 540 评估的具有高膜流动性的存活精子的百分比在 SLC 样本中低于对照组样本，与精液样本的初始精液特征无关。SLC 样本解冻后的存活精子产生的活性氧（用 CM-H（2）DCFDA 评估）低于对照组样本，尤其是在次标准精液样本中。这些发现表明，在冷冻前使用 SLC 对精子进行选择可提高猪精子的冷冻能力。
