Fleisch A, Malama E, Witschi U, Leiding C, Siuda M, Janett F, Bollwein H
Clinic of Reproductive Medicine, Vetsuisse-Faculty University of Zürich, Zürich, Switzerland.
Clinic of Reproductive Medicine, Vetsuisse-Faculty University of Zürich, Zürich, Switzerland.
Theriogenology. 2017 Feb;89:255-262. doi: 10.1016/j.theriogenology.2016.10.018. Epub 2016 Oct 28.
This study was designed to investigate the effects of an equilibration period up to 96 hours and three extenders (AndroMed, OPTIXcell, and Triladyl) on the quality of cryopreserved bull semen and to evaluate, whether an extension of the equilibration time to 72 hours does affect fertility in the field. One ejaculate of 17 bulls was collected and divided into three equal aliquots and diluted, respectively, with the three extenders. Each aliquot was again divided into five parts and equilibrated for 4, 24, 48, 72, and 96 hours before freezing in an automatic freezer. Sperm motility, plasma membrane and acrosome integrity (PMAI), and DNA fragmentation index (% DFI) were measured during equilibration. In addition to the parameters measured during equilibration, the percentage of viable sperm cells with high mitochondrial membrane potential (HMMP) was measured immediately after thawing, and after 3 hours of incubation at 37 °C. Sperm motility was assessed using CASA, and PMAI, HMMP, and % DFI were measured using flow cytometry. Equilibration time did affect all parameters before freezing (P < 0.01), and also the extender affected all parameters except HMMP (P < 0.05). After thawing, all parameters except HMMP immediately after thawing were influenced by the equilibration period (P < 0.001), whereas all parameters except % DFI immediately after thawing were influenced by the extender (P < 0.001). The changes of semen characteristics during 3 hours of incubation were also dependent on the equilibration time and the extender used in all parameters (P < 0.01). In the field study, semen of nine bulls was collected thrice weekly, processed using Triladyl egg yolk extender, and frozen in 0.25 mL straws with 15 × 10 spermatozoa per straw. In total, the nonreturn rates on Day 90 after insemination (NRR90) of 263,816 inseminations in two periods were evaluated. Whereas semen collected on Mondays and Wednesdays was equilibrated for 24 hours in both periods, semen collected on Fridays was equilibrated for 4 hours in period one and equilibrated for 72 hours in period 2. No differences in NRR90 could be found (P > 0.05). In conclusion, extension of the equilibration time from 4 hours to 24-72 hours can improve motility and viability of cryopreserved semen after thawing. The extent of improvement in semen quality is dependent on the extender used. Prolongation of the equilibration period from 4 hours to 72 hours had no effect on fertility in the field.
本研究旨在调查长达96小时的平衡期以及三种稀释剂（AndroMed、OPTIXcell和Triladyl）对冷冻保存的公牛精液质量的影响，并评估将平衡时间延长至72小时是否会影响实际生育能力。采集了17头公牛的一份射精样本，将其分成三个等量的部分，分别用三种稀释剂进行稀释。每个部分再分成五份，在自动冷冻仪中冷冻前分别平衡4、24、48、72和96小时。在平衡过程中测量精子活力、质膜和顶体完整性（PMAI）以及DNA碎片化指数（% DFI）。除了在平衡过程中测量的参数外，在解冻后以及在37°C孵育3小时后立即测量具有高线粒体膜电位（HMMP）的活精子细胞百分比。使用计算机辅助精子分析（CASA）评估精子活力，使用流式细胞术测量PMAI、HMMP和% DFI。平衡时间在冷冻前确实影响所有参数（P < 0.01），并且稀释剂也影响除HMMP之外的所有参数（P < 0.05）。解冻后，除解冻后立即测量的HMMP外，所有参数均受平衡期影响（P < 0.001），而除解冻后立即测量的% DFI外，所有参数均受稀释剂影响（P < 0.001）。在3小时孵育过程中精液特征的变化在所有参数上也取决于平衡时间和所用的稀释剂（P < 0.01）。在实际研究中，每周三次采集9头公牛的精液，使用Triladyl蛋黄稀释剂进行处理，并以每支0.25 mL细管、每管含15×10个精子的方式冷冻。总共评估了两个时期263,816次授精后第90天的未返情率（NRR90）。在两个时期中，周一和周三采集的精液均平衡24小时，而周五采集的精液在第一时期平衡4小时，在第二时期平衡72小时。未发现NRR90有差异（P > 0.05）。总之，将平衡时间从4小时延长至24 - 72小时可提高解冻后冷冻精液的活力和存活率。精液质量的改善程度取决于所用的稀释剂。将平衡期从4小时延长至72小时对实际生育能力没有影响。
