Capacitation and acrosome reaction in buffalo bull spermatozoa assessed bychlortetracycline and Pisum sativum agglutinin fluorescence assay

Studies on buffalo sperm capacitation have been limited because of the non- availability of a direct assay system. We describe two methods for detectin g the acrosomal status of buffalo spermatozoa, namely chlortetracycline (CT C) fluorescence assay and Pisum sativum agglutinin (FITC-PSA) stain. We als o test them under various treatment regimens and simultaneously standardize and calibrate them with transmission electron microscopy. An initial compa rison of three physiological media, such as Krebs-Ringer bicarbonate buffer , Tyrode solution and Brackett & Oliphant medium (having different calcium concentrations and osmolality) used for studying the capacitation of buffal o spermatozoa and assessed by CTC, FITC-PSA, Giemsa stain and TEM, revealed Brackett & Oliphant medium to be marginally better than the other two medi a. When stained with chlortetracycline, three distinct fluorescent patterns were visible in buffalo spermatozoa under capacitating conditions. These w ere 'F' with fluorescence in the post acrosomal region characteristic of un capacitated acrosome-intact cells; 'B' with fluorescence on the anterior po rtion of the sperm head and a dark band in the postacrosomal region, charac teristic of capacitated, acrosome intact cells and 'AR' with a fluorescent band on the posterior portion of the head, characteristic of acrosome-react ed cells. The FITC-PSA intensely labels the acrosomal region of acrosome in tact buffalo sperm. Acrosome reacted sperms had diminished acrosomal labell ing by both the probes used. Buffalo spermatozoa was not capacitated when c alcium was either omitted from the medium or chelated with EGTA. In the pre sence of Ca2+ ionophore, A23187, 68% at 4 h and 85% at 8 h completed the ac rosome reaction. Time course studies revealed a 4 h incubation period at 1. 71 mM Ca2+ concentration to be necessary before transformation of 'F' to 'B ' cells could take place. Spontaneous acrosome reaction induced at 6 and 8 h incubation of buffalo spermatozoa in I(RB medium resulted in conversion o f 'B' cells to 'AR' cells while 'F' cells remained unchanged. A simultaneou s evaluation of acrosome intact and acrosome-reacted cells using FITC-PSA, Giemsa and TEM gave results similar to examination by CTC stain. Both the a ssays are rapid, reproducible, reliable and they detect an increase or decr ease in physiological acrosome reactions. They thus can be used to study ef fects of calcium and prove to be good monitoring systems to identify buffal o sperm capacitation and acrosome reaction in individual buffalo bulls for fertility studies. (C) 2001 by Elsevier Science Inc.