Area of Biochemistry, INITRA, School of Veterinary Sciences, University of Buenos Aires, Buenos Aires, Argentina.
Theriogenology. 2012 Oct 15;78(7):1548-56. doi: 10.1016/j.theriogenology.2012.06.023. Epub 2012 Aug 25.
The objectives were to evaluate postthaw sperm quality and the response to an inducer of in vitro sperm capacitation in boar sperm, cryopreserved with (T) or without (C) α-tocopherol. Boar sperm frozen in 0.2-mL pellets were thawed and washed (W) or selected by three methods: Percoll discontinuous gradient (PS) or Sephadex (Sigma-Aldrich, St. Louis, MO, USA) (neutral [S] or with ion exchange [S+IO] columns). All separation methods enhanced sperm motility, plasma membrane integrity, and functionality and acrosome integrity for both C and T samples (P < 0.05). The best results were obtained with S and ionic Sephadex column. There was a decrease (P < 0.05) in capacitation-like changes in C samples separated with Sephadex (W: 19 ± 0.9%, PS: 22 ± 2.5%, S: 17 ± 1.2%, and S+IO: 17 ± 2.0%). Cryopreservation with α-tocopherol decreased (P < 0.05) the percentage of cryocapacitated sperm (W: 14 ± 0.7%, PS: 14 ± 1.0%, S: 13 ± 1.0%, and S+IO: 14 ± 0.9%) compared with C samples, without differences among selection techniques. Freezing with α-tocopherol and subsequent selection decreased lipid peroxidation (W: 20.79 ± 2.64 nmol thiobarbituric acid reactive substances (TBARS)/10(8) sperm; PS: 13.15 ± 2.39 nmol TBARS/10(8) sperm; S: 13.20 ± 2.18 nmol TBARS/10(8) sperm, and S+IO: 13.62 ± 2.76 nmol TBARS/10(8) sperm), with respect to washed and selected C samples (W: 37.69 ± 5.34 nmol TBARS/10(8) sperm, PS: 25.61 ± 5.85 nmol TBARS/10(8) sperm, S: 19.16 ± 3.28 nmol TBARS/10(8) sperm, and S+IO: 22.16 ± 6.09 nmol TBARS/10(8) sperm). In vitro capacitation levels were significantly higher for neutral Sephadex-selected T samples in comparison with C and unselected samples. These results were confirmed with a follicular fluid-induced acrosome reaction. In conclusion, cryopreserved sperm with α-tocopherol and subsequent Sephadex selection, improved postthaw quality and functionality of boar sperm, which could be useful for assisted reproductive techniques.
目的在于评估添加或不添加 α-生育酚（tocopherol）冷冻保护剂对猪精子冷冻后活力和体外获能反应的影响。将猪精液用 0.2ml 细管冷冻，解冻后洗涤（W）或通过三种方法进行选择：Percoll 不连续梯度（PS）或葡聚糖（Sigma-Aldrich，圣路易斯，密苏里州，美国）（中性[S]或带离子交换柱[S+IO]）。所有分离方法均能提高 C 和 T 样本的精子活力、质膜完整性和功能及顶体完整性（P < 0.05）。S 和离子交换柱分离效果最佳。Sephadex 分离的 C 样本获能样变化减少（P < 0.05）（W：19 ± 0.9%，PS：22 ± 2.5%，S：17 ± 1.2%，S+IO：17 ± 2.0%）。与 C 样本相比，添加 α-生育酚冷冻降低了冷冻获能精子的比例（W：14 ± 0.7%，PS：14 ± 1.0%，S：13 ± 1.0%，S+IO：14 ± 0.9%），但选择技术之间无差异。添加 α-生育酚冷冻和随后的选择降低了脂质过氧化（W：20.79 ± 2.64 nmol 硫代巴比妥酸反应物质（TBARS）/10（8）精子；PS：13.15 ± 2.39 nmol TBARS/10（8）精子；S：13.20 ± 2.18 nmol TBARS/10（8）精子，S+IO：13.62 ± 2.76 nmol TBARS/10（8）精子），与洗涤和选择的 C 样本相比（W：37.69 ± 5.34 nmol TBARS/10（8）精子，PS：25.61 ± 5.85 nmol TBARS/10（8）精子，S：19.16 ± 3.28 nmol TBARS/10（8）精子，S+IO：22.16 ± 6.09 nmol TBARS/10（8）精子）。与 C 样本和未选择样本相比，添加 α-生育酚且经 Sephadex 选择的 T 样本体外获能水平显著提高。用卵泡液诱导的顶体反应证实了这一结果。总之，添加 α-生育酚并随后用 Sephadex 选择可改善猪精子冷冻后的活力和功能，这可能对辅助生殖技术有用。
