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

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.