Short communication. Evaluation of a commercial kit based on acridine orange/propidium iodide to assess the plasma membrane integrity of ram sperm

This study was designed to develop a semiautomatic computer assisted methodology to evaluate the membrane integrity of ram spermatozoa using a commercial kit based on acridine orange/propidium iodide (AO/PI) labelling and ImageJ software. The study was divided into two experiments. In the first trial, the new computer-assisted method was validated by mixing fresh semen samples with different volumes of freeze killed spermatozoa to determine proportions of damaged spermatozoa in the final samples. The proportion of damaged spermatozoa in each sample determined by the automated procedure where highly correlated (R2=0.97, p<0.001) with the predicted theoretical values. In the second trial, the new method was compared with a previously validated method of membrane integrity assessment based on phase-contrast/propidium iodide (PH/PI) methodology. Measurements by AO/PI were, on average, 4.0% larger than measurements by PH/PI (SD=7.02%) and 1.79% smaller than measurements of sperm motility determined by CASA (SD=4.83). The AO/PI method was also more repeatable than the PH/PI. The double staining methodology coupled with the routine for image analysis allowing automatic determination of sperm membrane integrity means a reduction in processing time of 75% compared to the previously developed method using a single fluorochrome (3 vs 12 min on average if the incubation period was included). This facilitates its use when a large number of samples are analysed. Our results validate the new computer assisted method for assessing sperm membrane integrity in sheep. The new method developed, in addition to being a free tool, allows quick automatic determination of sperm viability, which facilitates its use in routine semen analysis