title: Extracellular calcium is involved in egg yolk-induced head-to-head agglutination of bull sperm.
abstract: Head-to-head agglutination of bull sperm occurs when semen is highly diluted in an egg yolk-citrate diluent without streptomycin. The objectives were to investigate causes of sperm agglutination and the underlying mechanism. Aliquots of bull semen were diluted in a base diluent (BD) supplemented with various test components and the percentage of agglutinated sperm (% AggSp) was quantified at 1, 5, 24, 48, and 72 h of incubation. When sperm were incubated at 22 °C, no agglutination was observed in BD for up to 72 h, whereas the % AggSp was 5.0, 41.7, 72.2, 91.1, and 92.8% in BD + 5% egg yolk (BD + EY) at 1, 5, 24, 48 and 72 h, respectively. However, no sperm agglutination was observed in BD + EY if incubation temperature was 37 °C. Addition of 5 or 10 mM ethylenebis (oxyethyleneni-trilo) tetra-acetic acid to BD + EY reduced the % AggSp from 95% to <5% at 72 h (P < 0.001), but addition of 5 mM CaCl(2) to BD failed to induce sperm agglutination in the absence of egg yolk, implicating calcium and other factors in egg yolk. Addition of the citrate-soluble fraction (CSF) of egg yolk to BD induced sperm agglutination similar to whole egg yolk, whereas water- and saline-soluble fractions of egg yolk were ineffective. The sperm-agglutinating efficacy of CSF (the % AggSp = 95% at 72 h) was reduced by dialysis (20%; P < 0.05), partially restored by addition of 5 mM CaCl2 (70%; P < 0.05), but the calcium effect was neutralized by addition of 5 mM ethylenebis (oxyethyleneni-trilo) tetra-acetic acid (1.7%; P < 0.05), again implicating calcium. Addition of 30 μM of a protein kinase A inhibitor (H-89) to an agglutinating diluent failed to inhibit sperm agglutination, whereas addition of 2 mM of a cAMP analogue, dbcAMP, to a nonagglutinating diluent failed to induce sperm agglutination. Agglutination status had no effect on sperm plasma membrane/acrosome status and mitochondrial membrane potential. In conclusion, calcium and other component(s) in the CSF of egg yolk induced head-to-head agglutination of bull sperm in a time- and temperature-dependent manner. Although the mechanism of agglutination was not determined, the cAMP- protein kinase A signaling pathway was not involved.
authors.affiliation: Livestock Improvement Corporation, Hamilton, New Zealand. dyang@lic.co.nz
authors.forename: D H
authors.initials: DH
authors.lastname: Yang
authors.forename: A G
authors.initials: AG
authors.lastname: McMillan
authors.forename: N T
authors.initials: NT
authors.lastname: Standley
authors.forename: P
authors.initials: P
authors.lastname: Shannon
authors.forename: Z Z
authors.initials: ZZ
authors.lastname: Xu
mesh_terms: D000177:Acrosome / Q000502:physiology; D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D002122:Calcium Chloride / Q000494:pharmacology; D002417:Cattle; D002462:Cell Membrane / Q000502:physiology; D019343:Citric Acid; D004530:Egg Yolk / Q000737:chemistry; D004533:Egtazic Acid / Q000494:pharmacology; D008297:Male; D053078:Membrane Potential, Mitochondrial / Q000502:physiology; D012995:Solubility; D013073:Sperm Agglutination / Q000187:drug effects*; D013094:Spermatozoa / Q000502:physiology* / Q000648:ultrastructure; D013696:Temperature
chemical_list: D019343:Citric Acid; D004533:Egtazic Acid; D002122:Calcium Chloride; D002118:Calcium