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Title: Comparison of effects of cryopreservation diluents on the ability of Ram sperm to reduce resazurin dye

item WONDERLY, JESSICA - University Of Wyoming
item KOEPKE, KALLI - University Of Wyoming
item ALEXANDER, BRENDA - University Of Wyoming
item Purdy, Phil

Submitted to: Research Day Abstracts: Regional Universities Research Day
Publication Type: Abstract Only
Publication Acceptance Date: 4/7/2017
Publication Date: 4/28/2017
Citation: Wonderly, J.L., Koepke, K., Alexander, B., Purdy, P.H. 2017. Comparison of effects of cryopreservation diluents on the ability of Ram sperm to reduce resazurin dye. Research Day Abstracts: Regional Universities Research Day.

Interpretive Summary:

Technical Abstract: Resazurin dye is an effective way to test the metabolism of sperm. As sperm move, they create metabolic waste which is detected by the dye. Another way sperm are evaluated is by Computer-Assisted Sperm Analysis (CASA). CASA detects motility, progression, curvilinear velocity, lateral head amplitude, beat cross frequency, and size. For this experiment, sperm characteristics were evaluated and compared across semen extenders using CASA and resazurin reduction test. Semen from five rams was collected in January and frozen in liquid nitrogen. Individual ram samples were cryopreserved using either 300 mM Tris egg yolk glycerol (TEYG), 200 mM TEYG, or skim milk egg yolk glycerol (SMEY) extenders. After thawing, sperm from each diluent was analyzed using CASA at 0 and 15 minutes. Sperm metabolic activity was determined using 22 mg of resazurin per 100 mL of diluent and combined with an additional100 µL of diluent. Samples were incubated at 45°C for 30 minutes and observed for color change indicating metabolic activity. Sperm frozen in 200 or 300 mM Tris had higher (P=0.04) metabolic activity after thawing than sperm preserved in skim milk. Sperm motility was similar (P>0.05) at thaw regardless of the extender, but curvilinear velocity was greater (P=0.04) in sperm extended in 200 or 300 mM Tris at 15 min. Sperm head movement was greater (P<0.01) for samples frozen with 200 mM Tris extender. Using stepwise regression to predict time for color change in the resazurin test, at initial testing (time=0), progression and head size remained in the model (p = 0.002) accounting for ~70% of the variability. However, after 15 minute incubation, curvilinear velocity was the only factor (P=0.003) in the regression, explaining 45% of the variability. At initial testing, progression (P=0.011; r2=-0.63) and curvilinear velocity (P=0.033; r2=-0.55) were negatively correlated to time to color change for the resazurin dye test. After 15 minutes of incubation, curvilinear velocity (P=0.003; r2=-0.7) and lateral head amplitude (P=0.050; r2=-0.51) were the only correlated variables. Motility tended (P=0.09) to be associated with resazurin results at initial testing and at 15 minutes. Type of cryopreservation diluent used influences the metabolic activity of sperm at thaw. Resazurin dye test could be used as a predictor for progression at time. Evaluation of the frozen-thawed semen indicated that 200 mM Tris is an acceptable extender supporting increased metabolic activity and motility.