PARENT SESSION
Capacitation & Acrosome Reaction

EFFECT OF THAWING, CAPACITATING, AND CULTURE PROCEDURES ON STALLION SPERM MEMBRANE INTEGRITY AND CAPACITATION STATUS.

Gandolfi, Fulvio¹, Colleoni, Silvia ¹, Luciano, Alberto¹, Modina, Silvia¹, ¹

ABSTRACT- The use of IVF in horse has a limited efficency reflecting low oocyte developmental competence and inadequate sperm capacitation procedures. Aim of this study was to investigate the effect of thawing, capacitation and culture procedures on frozen horse spermatozoa. Spermatozoa were thawed in water, at 37° C, and layered on top of a 45-90% Percoll gradient made with TALP medium and centrifuged for 30 minutes at 600 x g. The sperm pellet was washed once in the same medium, counted and diluted to a final concentration of 1 x 10⁶ spermatozoa/ml TALP supplemented with 0.6% (w/v) BSA fatty acid free and 12 g/ml heparin (TALP-IVF). Sperm cells were incubated with 0, 2, 4 or 8 in vitro matured cumulus-oocyte complexes (COC). The combination of carboxyfluorescein diacetate and propidium iodide was used to evaluate sperm membrane integrity and the chlortetracycline-assay to determine the capacitation status of spermatozoa. Sperm cells were examined after thawing, 0, 2 and 18 h from the beginning of incubation in TALP-IVF. Each experiment was replicated at least 3 times. The freezing-thawing procedure left only 56.6 ± 3.4 % of the sperm cells with an intact membrane but most of them (90.04 ± 0.4%) were non-capacitated and with an intact acrosome. The following incubation in TALP-IVF alone induced membrane damage at high rates with only 9.58 ± 1.8 % of them intact after 18h. However the presence of COC in the medium significatively increased the number of membrane-intact spermatozoa at the end of incubation and 2 COC had a lower effect than 4 or 8 (35.48 ± 4.21% vs. 50.39 ± 4.17% and 53.87 ± 1.99%, respectively). Incubation in TALP-IVF with or without COC induced a low rate of sperm capacitation even after 18 h (5.22 ± 0.6% and 8.6 ± 0.4%, respectively). We conclude that the sperm thawing and capacitating procedures used in this experiment can damage the cell membrane but the presence of 4 or more COC in TALP-IVF could prevent further damages. COC, however, were unable to induce the acrosome reaction at high rates.

KEY WORDS: stallion, IVF, sperm preparation