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Title: VALIDATION OF A RAPID, LARGE-SCALE ASSAY TO QUANTIFY ATP LEVELS IN SPERMATOZOA

Author
item Long, Julie
item Guthrie, Howard

Submitted to: Theriogenology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/2/2005
Publication Date: 5/10/2006
Citation: Long, J.A., Guthrie, H.D. 2006. Validation of a rapid, large-scale assay to quantify atp levels in spermatozoa. Theriogenology. 65:1620-1630.

Interpretive Summary: Experiments involving assessment of sperm function often require measurement of a large number of samples. For example, determining the effect of alternative extenders for short-term storage of turkey sperm requires a high sampling frequency from multiple groups during the 24h storage period. We found that individual assessment of samples for ATP level did not support such expansive experimental designs. After unsuccessfully attempting to reproduce the only published method for batch analysis of ATP in sperm (using a scintillation counter), we developed an alternative method using a plate reader and defined the assay conditions to produce highly repeatable results. The ATP assay was validated for 3 species (turkey, rooster, pig), using both fresh semen and species-specific semen storage protocols.

Technical Abstract: 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 3 different species, including turkey, rooster and boar sperm. The sensitivity of the assay differed between avian and mammalian sperm, with 2.5x106 sperm being the lowest number of turkey and rooster sperm that could be assayed compared to 2.5x105 boar sperm. ATP levels in fresh turkey semen ranged from 4.14 to 15.6 nmol/109 sperm; similarly, freshly collected rooster semen contained from 2.16 to 21.4 nmol ATP/109 sperm. Evaluation of turkey semen that had been stored at 4 C for 24h revealed a decline in ATP levels (2.35 ± 0.34 nmol ATP/109 sperm). Likewise, cryopreserved rooster sperm contained lower levels of ATP (48.1 ± 1.56 pmol ATP/109 sperm) than non-stored sperm. Boar sperm contained similar levels of ATP, whether fresh (74.2 ± 8.1 pmol ATP/106 sperm), stored for 1 day (77.0 ± 8.1 pmol ATP/106 sperm) or 5 days (81.96 ± 8.1 pmol ATP/106 sperm). For all 3 species, assay variation was low (inter-assay, 0.66-1.9% CV; intra-assay, 1.3% CV).