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Resazurin Reduction Assay to Evaluate the Quality of Thawed Cryopreserved Stallion Spermatozoa Motility.

机译：刃天青还原还原法评估解冻的冷冻保存的种马精子活动性的质量。

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摘要

The purpose of this research was to determine if the fertility potential of thawed cryopreserved stallion spermatozoa could be assessed using the resazurin reduction assay. Post cryopreservation, thawed stallion sperm motility will vary greatly, and as a result so will the amount of nicotinamide adenine dinucleotide dehydrogenase (NADH) present that is being produced by motile sperm cells. In the presence of NADH the reducible dye resazurin will change from a blue color, to a pink (resorufin), and finally to a colorless (dihydroresorufin) state. A higher percentage of post-thaw motile cells may result in a more rapid time of dye reduction. Therefore, resazurin may act as an indicator for mitochondrial activity and motility of thawed samples. 30 ejaculates were collected from 2 Quarter horse stallions using a Missouri model artificial vagina. Gel-free samples were assessed for concentration, motility and volume; then diluted and centrifuged. The sperm pellet was split and diluted with either Lactose EDTA (LE) cryopreservation extender or Modified French Formula 5 (MFR5) extender to a concentration of 200 x 106 sperm per mL and cooled to 5° C. Samples were split into controls (no resazurin) and treatments (0.5 muL of a 6.77 muM resazurin solution/mL) for both extenders. Samples were packed in French polyvinyl 0.5 mL straws at a concentration of 200 x 106 per mL and cryopreserved using liquid nitrogen. Control and treatment samples, processed with either LE extender or MFR5 extender, were thawed in a 37° C water bath and observed until a color change was evident, or up to 20 min. Additional assisted semen analysis (CASA) and flow cytometric analyses (live -- mitochondria, acrosome intact or acrosome reacted) were implemented to assess dye toxicity. Diagnostic statistics were performed, with the parameter of ≥ 30% motility considered to be adequate fertility potential, for determination of the validity of the assay. There were no significant differences (P ≤ 0.05) between the control and treatment groups for any of the CASA or flow cytometric parameters indicating the absence of dye toxicity. Likewise, there were no significant differences in CASA and flow cytometric parameters between samples processed with the different extenders with the exception of LE extended samples (3.9 +/- 3.5) having a significantly (P ≤ 0.05) greater percentage of progressively motile spermatozoa than the MFR5 extended samples (0.9 +/- 1.5). Sensitivity is the percentage of known low fertility potential samples (< 30% motility) that did not reduce the dye within a set time interval while specificity is defined as the percentage of known high fertility samples (≥ 30% motility) that reduced the dye within a set time interval. The highest sensitivity was 68% at 8 min for the MFR5 extender and 98% at 18 min for the LE extender. The greatest specificity was 82% for the MFR5 extender and 80% for the LE extender at one min. The Positive Predictive Value (PPV) represents the percentage of unknown low fertility samples (< 30% motility) that would not reduce the dye within a set time interval while the Negative Predictive Value (NPV) illustrates the proportion of unknown high fertility (≥ 30% motility) samples that reduced the dye within a certain time interval. The MFR5 extender had a PPV of 22% at 8 min and the LE extender a PPV of 82% at 6 min. The MFR5 extender had a NPV of 90% at 8 min and the LE extender a NPV of 48% at 13 min. The overall accuracy for the MFR5 extender ranged from 15% to 79%, peaking at minutes 1 to 3. The overall accuracy for the test on LE extender ranged from 30% to 75%, peaking at min 13 to 15. These results suggest that the resazurin reduction assay is a nontoxic test that can be used to determine the fertility potential of stallion spermatozoa cryopreserved using either MFR5 or LE extender.
机译：这项研究的目的是确定是否可以使用刃天青素还原试验评估解冻的低温保存的种马精子的受精潜力。冷冻保存后，解冻的种马精子活力会发生很大变化，结果，运动型精子细胞产生的烟酰胺腺嘌呤二核苷酸脱氢酶（NADH）的数量也会随之变化。在存在NADH的情况下，还原型刃天青素将从蓝色变为粉色（间苯二酚），最后变为无色（二氢间苯二酚）状态。融化后的运动细胞百分比较高，可能导致染料还原的时间更快。因此，刃天青可以作为解冻样品的线粒体活性和运动性的指标。使用密苏里州模型的人工阴道从2个四分之一的公马中收集了30份射精。评估无凝胶样品的浓度，运动性和体积。然后稀释并离心。分离精子沉淀，并用乳糖EDTA（LE）冷冻保存增量剂或改良的French Formula 5（MFR5）增量剂稀释至每毫升200 x 106精子的浓度，冷却至5°C。将样品拆分为对照（无刃天青）和两种扩展剂的处理方法（0.5μL的6.77μM刃天青溶液/ mL）。将样品包装在浓度为200 x 106 / mL的法国聚乙烯0.5 mL吸管中，并使用液氮冷冻保存。将用LE补充剂或MFR5补充剂处理的对照和处理样品在37°C水浴中融化，观察直至明显的颜色变化，或长达20分钟。另外还进行了辅助精液分析（CASA）和流式细胞仪分析（活的线粒体，完整的顶体或顶体反应）来评估染料毒性。进行诊断统计，并以≥30％的运动性参数作为足够的生育力，以确定测定的有效性。对照组和治疗组之间的任何CASA或流式细胞仪参数均无显着差异（P≤0.05），表明不存在染料毒性。同样，在不同扩展剂处理的样品之间，CASA和流式细胞仪参数也无显着差异，除了LE扩展样品（3.9 +/- 3.5）的活动性精子百分比显着高于（P≤0.05）。 MFR5扩展样本（0.9 +/- 1.5）。灵敏度是在设定的时间间隔内未还原染料的已知低生育力样本（<30％活力）的百分比，而特异性则定义为在其中降低染料的已知高生育力样本（≥30％活力）的百分比设定的时间间隔。 MFR5补充剂在8分钟时的最高灵敏度为68％，LE补充剂在18分钟时的最高灵敏度为98％。一分钟时，MFR5延伸剂的最大特异性为82％，LE延伸剂的最大特异性为80％。正预测值（PPV）表示在设定的时间间隔内不会还原染料的未知低繁殖力样品（<30％活力）的百分比，而负预测值（NPV）则表示未知高繁殖力（≥30）的比例在一定时间间隔内还原染料的样品。 MFR5补充剂在8分钟时的PPV为22％，LE补充剂在6分钟时的PPV为82％。 MFR5补充剂在8分钟时的NPV为90％，LE补充剂在13分钟时的NPV为48％。 MFR5扩展剂的总体准确度为15％至79％，在第1至3分钟达到峰值。LE扩展剂的测试总体准确性为30％至75％，在13至15分钟达到峰值。这些结果表明，刃天青素还原试验是一种无毒试验，可用于确定使用MFR5或LE补充剂冷冻保存的种马精子的受精潜力。

著录项

  • 作者

    Genetti, Melissa Ann.;

  • 作者单位

    Sul Ross State University.;

  • 授予单位 Sul Ross State University.;
  • 学科 Agriculture General.;Biology General.
  • 学位 M.S.
  • 年度 2013
  • 页码 64 p.
  • 总页数 64
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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