Which fields under a coverslip should one assess to estimate sperm motility?

Under field conditions the motility of bull semen often has to be estimated under a coverslip on a microscope slide. This study was aimed at determining which combination of fields under coverslips provides measurements of sperm motility that best represent the motility in semen specimens as measured in a specially designed chamber for use in a computer-assisted sperm analyzer (CASA). We measured the motility (percentages motile, progressively motile, and aberrantly motile spermatozoa) in each of four straws of frozen-thawed semen from each of 10 bulls five times, ranging from 5 to 120 minutes after thawing with each bull by straw by time combination yielding one semen specimen. Motility was measured in duplicate in a Hamilton, Thorne IVOS CASA; once in each of 12 fields equally spaced along the equatorial radius of a coverslip (Field 0 at the edge and Field 11 at the center) and once in each of eight equally spaced fields along the equator of a Leja 4 chamber designed for use in a CASA. We used the weighted average motility of all fields in a chamber as gold standard and compared it to the average motility of each the following combinations of fields under the coverslip: all 12 fields, Fields 2 to 4, Fields 2 and four, Field 3 and the center three fields. The concordance correlation coefficient (CCC) was determined between the motility in each combination of fields under coverslips and the chambers as a reproducibility index, which evaluates the agreement between the readings under the coverslips and the gold standard readings in the chambers (n = 187 for each CCC). We performed pairwise comparisons of the CCCs (P < 0.005 for each comparison) and established that the average motility under all 12 fields better reproduced the motility in the chamber than the center three fields or Field 3. The averages of Fields 2 to 4 and Fields 2 and 4 reproduced chamber motility as well as the average of all 12 fields, except for the percentage motile sperm, where the average of all 12 fields was better. Using the average motility of Fields 2 and 4, 50% of estimates fell within 6%, 4% and 3% above or below the percentages motile, progressively motile and aberrantly motile spermatozoa in the Leja 4 chamber, 80% of estimates fell within 12%, 8% and 7% thereof and 95% fell within 23%, 13% and 12% thereof. In conclusion, for the method of spreading semen under a coverslip and the range in motility values used, this study shows that the average of the motility over the 12 fields along the equatorial radius under a coverslip provides the best estimate of the motility of a semen specimen, while the average of Fields 2 and 4 is also suitable for the subjective estimation of motility under field conditions, although the estimated motility is expected to fall within 6% above or below the motility of the specimen in only 50% of semen specimens.