Wrocław University of Environmental and Life Sciences, Faculty of Veterinary Medicine, Department of Reproduction and Clinic of Farm Animals, Wrocław, Poland.
Theriogenology. 2011 Sep 15;76(5):843-50. doi: 10.1016/j.theriogenology.2011.04.016. Epub 2011 Jun 12.
The present study was conducted to investigate spermatozoal membrane integrity, acrosome integrity, mitochondrial activity, and chromatin structure in fresh and frozen-thawed Canada goose (Branta canadensis) semen with the use of the flow cytometry. The experiment was carried out on ten, 2-year-old, Canada goose ganders. The semen was collected twice a week, by a dorso-abdominal massage method, then pooled and subjected to cryopreservation in straws, in a programmable freezing unit with the use of dimethyloformamide (DMF) as a cryoprotectant. Frozen samples were thawed in a water bath at 60 °C. The freezing procedure was performed ten times. For the cytometric analysis the fresh and the frozen-thawed semen was extended with EK extender to a final concentration of 50 million spermatozoa per mL. Sperm membrane integrity was assessed with SYBR-14 and propidium iodide (PI), acrosomal damage was evaluated with the use of PNA-Alexa Fluor®488 conjugate, mitochondrial activity was estimated with Rhodamine 123 (R123), and spermatozoal DNA integrity was measured by the sperm chromatin structure assay (SCSA). The cryopreservation of Canada goose semen significantly decreased the percentage of live cells, from 76.3 to 50.4% (P < 0.01). Moreover, we observed the significant decrease in the percentage of live spermatozoa with intact acrosomes (P < 0.01), but we did not detect significant changes in the percentage of live spermatozoa with ruptured acrosomes. However, after thawing 50% of Canada goose live spermatozoa retained intact acrosomes. Furthermore, the percentage of live spermatozoa with active mitochondria was significantly lower in the frozen-thawed semen than in the fresh semen (P < 0.05). Nevertheless, after thawing the mitochondria remained active in almost 50% of live cells. In the present study, we observed no changes in the percentage of sperm with fragmented DNA after freezing-thawing of Canada goose semen. In conclusion, the present study indicates that even the fresh Branta canadensis semen might have poor quality, the cryopreservation of its semen did not provoke spermatozoal DNA defragmentation and half of the spermatozoa retained intact acrosomes and active mitochondria after freezing-thawing.
本研究旨在使用流式细胞术研究新鲜和冷冻解冻加拿大鹅（Branta canadensis）精液中的精子膜完整性、顶体完整性、线粒体活性和染色质结构。实验在十只 2 岁的加拿大公鹅身上进行。每周通过背-腹部按摩法采集两次精液，混合后在可编程冷冻装置中使用二甲基甲酰胺（DMF）作为冷冻保护剂在 straw 中进行冷冻保存。冷冻样品在 60°C 的水浴中解冻。冷冻程序进行了十次。对于细胞分析，将新鲜和冷冻解冻的精液用 EK extender 扩展至最终浓度为每毫升 5000 万个精子。使用 SYBR-14 和碘化丙啶（PI）评估精子膜完整性，使用 PNA-Alexa Fluor®488 缀合物评估顶体损伤，使用 Rhodamine 123（R123）估计线粒体活性，通过精子染色质结构分析（SCSA）测量精子 DNA 完整性。加拿大鹅精液的冷冻保存显著降低了活细胞的百分比，从 76.3%降至 50.4%（P < 0.01）。此外，我们观察到具有完整顶体的活精子的百分比显著降低（P < 0.01），但未检测到具有破裂顶体的活精子的百分比有显著变化。然而，解冻后，50%的加拿大鹅活精子保留完整的顶体。此外，冷冻解冻精液中具有活性线粒体的活精子的百分比明显低于新鲜精液（P < 0.05）。然而，解冻后，近 50%的活细胞中线粒体仍然活跃。在本研究中，我们观察到冷冻解冻加拿大鹅精液后 DNA 碎片化的精子百分比没有变化。总之，本研究表明，即使是新鲜的加拿大鹅精液也可能质量较差，其精液的冷冻保存不会引起精子 DNA 碎片化，并且冷冻解冻后一半的精子保留完整的顶体和活性线粒体。
