Validation of a rapid, large-scale assay to quantify ATP concentration in spermatozoa

Quantification of ATP content in spermatozoa is a useful assay for evaluating sperm function; however, most detection methodology relies on assessing single samples. We have developed and validated a highly repeatable assay that permits simultaneous measurement of up to 78 samples. A key feature of this assay includes combination of a phosphatase inhibition and ATP extraction step that permits maximal detection of ATP and sample storage at −20 °C prior to assay. The assay was validated for spermatozoa from three different species, including turkey, rooster and boar. The sensitivity of the assay differed between avian and mammalian spermatozoa, with 2.5 × 106 spermatozoa being the lowest number of turkey and rooster spermatozoa that could be assayed compared to 2.5 × 105 boar spermatozoa. Concentrations of ATP in fresh turkey semen ranged from 2.14 to 15.6 nmol/109 spermatozoa; similarly, freshly collected rooster semen contained from 2.16 to 21.4 nmol ATP/109 spermatozoa. Evaluation of turkey semen that had been stored at 4 °C for 24 h revealed a decline in ATP concentrations (2.35 ± 0.34 nmol ATP/109 spermatozoa). Likewise, cryopreserved rooster spermatozoa contained lower concentrations of ATP (0.05 ± 0.01 nmol ATP/109 spermatozoa) than non-stored spermatozoa. Boar spermatozoa contained similar concentrations of ATP, whether fresh (74.2 ± 8.1 pmol ATP/106 spermatozoa), stored for 1 day (77.0 ± 8.1 pmol ATP/106 spermatozoa) or 5 days (81.96 ± 8.1 pmol ATP/106 spermatozoa). For all three species, assay variation was low (inter-assay, 0.66–1.9% CV; intra-assay, 1.3% CV).