Effects of calcium and bicarbonate on head-to-head agglutination in ejaculated boar spermatozoa

The present study was conducted to reveal the effects of calcium and bicarbonate on the occurrence of head-to-head agglutination in ejaculated boar spermatozoa in vitro. Boar spermatozoa were washed and incubated in a modified Krebs-Ringer bicarbonate (mKRB) in a 37 degrees C CO2 incubator (5% CO2 in air) for 1-5 h. Before and after the incubation, aliquots of each sperm sample were fixed, smeared on glass slides, and stained with a phosphate-buffered solution of Giemsa to assess the percentages of head-to-head agglutinated spermatozoa. Before the incubation, only 5-12% of the spermatozoa were agglutinated. After the 1-h incubation, however, the percentage of head-to-head agglutinated spermatozoa rose to approximately 50%, followed by only minor increases thereafter. This rise was dependent on the concentrations of calcium chloride contained in the mKRB and was attenuated by the addition of 2 mM [ethylenebis(oxyethylenenitrilo)]tetra-acetic acid (EGTA) to the medium. Moreover, the replacement of sodium bicarbonate with 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (Hepes) in the medium and treatment with ruthenium red, which have both been shown previously to inhibit calcium uptake by boar spermatozoa, significantly reduced the rise. Based on these findings, it was concluded that extracellular calcium and bicarbonate are key factors regulating head-to-head agglutination in boar spermatozoa. The possible relationship between agglutinability and the fertilizing ability of boar spermatozoa is also discussed.