Crude mare follicular fluid exerts chemotactic effects on stallion spermatozoa

Ejaculates from two stallions (7 and 17 years of age) were used for studying possible chemotactic effects of crude mare follicular fluid in vitro. Five millilitres of fresh semen samples collected at a horse ranch were diluted in 5 mi of HTF (Human Tubal Fluid) medium and transported to the laboratory facilities, keeping the temperature at 37 degrees C. Sperm washing was carried out in 1 mi aliquots of these specimens, diluted 1 : 1 (v:v) in HTF medium added with 10% HSA (HTF-HSA) and centrifuged at 400 x g. Pellets were resuspended in 1 mi of HTF-HSA and sperm concentration adjusted to 50 million/ml for the assays. Ten-microlitre aliquots of washed semen samples were placed in a plastic capillary connecting two equidistant chambers filled either with crude follicular fluid or a control fluid (HTF-HSA), and incubated for different lengths of time (0-30 min) at room temperature. At the end of the incubation period, fluids from both chambers were withdrawn separately and placed in 12 mm x 75 mm plastic tubes. The tubes were then centrifuged at 1500 x g and the pellets were resuspended in 0.3 mi of HTF-HSA. Sperm that migrated to the chambers were counted in 10 mu l aliquots on a hemocytometer and specific and nonspecific migration was calculated. In a total of 48 experiments, the mean percentage of sperm migration to the follicular fluid from mares was higher than that found on the control chambers (66.05 +/- 3.29% versus 14.35 +/- 10.94) and the acme of specific migration (78.14%) was reached at 30 min. These experiments settle the conditions for further assays in order to disclose the chemical nature of putative chemoattractants present in follicular fluid of mares and suggest that sperm chemotaxis may also occur in vivo in this particular species.