Suppression of PMN-chemotaxis by different molecular weight fractions of equine seminal plasma

The objective of this study was to determine the molecular weight of the factors in equine seminal plasma that suppress chemotaxis of polymorphonuclear neutrophils (PMNs). Blood plasma was pooled from four healthy horses and seminal plasma was pooled from two fertile stallions. Both the blood plasma and the seminal plasma were stored at -20°C until used. Complement in blood plasma was activated with E. Coli lipopolysaccharide (LPS) in the presence of 2%, 10%, 20%, and 30% seminal plasma. In addition, blood plasma and heat inactivated blood plasma without seminal plasma were used as controls. An aliquot chosen to standardize the amount of blood plasma was diluted with McCoy's medium into a chemotactic chamber. Chemotaxis of blood-derived equine PMNs toward the chemoattractants was determined after incubation at 37°C for 45 minutes. Results were expressed as the percentages of positive controls (LPS-activated blood plasma in the absence of seminal plasma), and the mean of each level of seminal plasma was compared using the Statistical Analysis System, general linear model, and Duncan's multiple range test. Significance was set at P < 0.05. Seminal plasma inhibited PMN-chemotaxis in a dose dependent manner (P < 0.05). PMN-chemotaxis was more suppressed by seminal plasma than by heat inactivated blood plasma (P < 0.05). In a second experiment, seminal plasma proteins were separated in three different molecular weight fractions (< 10,000, < 50,000, and < 100,000 MW) by the use of Centricon ultracentrifugation (Amicon Inc.). Complement in blood plasma was activated with E. Coli LPS in the presence of 10% seminal plasma fractions with a molecular cut off of 10,000, 50,000, and 100,000 MW. All seminal plasma fractions suppressed PMN-chemotaxis (P < 0.05). The most marked chemotaxis suppression was found in the 50,000-100,000 MW fraction. Although heat inactivation of blood plasma caused a suppression of chemotaxis, this suppression was less than that caused by all seminal plasma fractions (P < 0.05). It was concluded that more than one macromolecule in seminal plasma suppresses PMN-chemotaxis. Although inactivation of complement is involved in this mechanism, additional factors may contribute to the observed suppression.