Adaptation of the hypoosmotic swelling test to assess functional integrity of stallion spermatozoal plasma membranes

• Published in: Theriogenology 2001-03, Vol.55 (4), p.1005-1018
• Main Authors: Nie, G.J., Wenzel, J.G.W.
• Format: Article
• Language: English
• Subjects: Animals; Cell Membrane - physiology; Cell Size; equine; Horses - physiology; HOS test; hypoosmotic swelling; Hypotonic Solutions; Male; Reproducibility of Results; Sensitivity and Specificity; Sperm Count; Spermatozoa - ultrastructure; spermatozoal plasma membrane; stallion; Temperature; Time Factors
• ISSN: 0093-691X; 1879-3231
• DOI: 10.1016/S0093-691X(01)00461-7

Hypoosmotic swelling (HOS) is used for assessing plasma membrane function and fertilizing capacity of human spermatozoa. However, HOS solutions and methodologies have not been evaluated specifically for assessing stallion spermatozoa. The objective of this study was to identify a HOS solution and assay conditions specifically for stallions that would maximize spermatozoal plasma membrane swelling. The HOS solutions and assay conditions, including incubation time (15 to 180 min), temperature (25° vs 37°C), and total number of cells examined (100, 200 or 500) were evaluated. Assay consistency, accuracy, reliability and repeatability also were determined. Maximum spermatozoal plasma membrane swelling was observed in a 100 mosmol sucrose solution (P < 0.001). Incubation time (P = 0.67), temperature (P = 0.70) and total number of spermatozoa examined (P = 0.38 and P = 0.24 for 100 vs 200 and 100 vs 500, respectively) did not influence percent of HOS positive spermatozoa observed. A high degree of assay accuracy was indicated when a correlation of r = 0.998 was obtained between the HOS positive spermatozoa observed and expected when known amounts of heat-treated spermatozoa, unable to undergo swelling, were added to untreated spermatozoa. Assay consistency was demonstrated, as reflected by a mean coefficient of variation of 0.073 for 4 stallions. Also, the coefficient of variation from 2 analyses of variance was 0.168 and 0.096, indicating reasonably good assay reliability; estimates of repeatability from the same analyses were 0.794 and 0.968. The HOS test adapted to stallion spermatozoa in this study is a simple, highly accurate and consistent assay with good reliability and repeatability. Results observed under the conditions evaluated also permit some flexibility in adapting this assay to individual laboratory and practice settings for evaluating stallion spermatozoal plasma membranes.