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

J. L. Yániz, I. Palacín, S. Vicente-Fiel, J. Gosalvez, C. López-Fernández, P. Santolaria

Abstract


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.

Keywords


fluorescence microscopy; Ovis aries; sperm quality; sperm viability

Full Text:

PDF

References


Alm K, Taponen J, Dahlbom M, Tuunainen E, Koskinen E, Andersson M, 2001. A novel automated fluorometric assay to evaluate sperm viability and fertility in dairy bulls. Theriogenology 56: 677-684.
http://dx.doi.org/10.1016/S0093-691X(01)00599-4 

Bland JM, Altman DG, 1986. Statistical methods for assessing agreement between two methods of clinical measurement. Lancet 1: 307-310.
http://dx.doi.org/10.1016/S0140-6736(86)90837-8 

Brito LFC, Barth AD, Bilodeau, GoeseelsS, Panich PL, Kastelic JP, 2003. Comparison of methods to evaluate the plasmalemma of bovine sperm and their relationship with in vitro fertilization rate. Theriogenology 60: 1539-1551.
http://dx.doi.org/10.1016/S0093-691X(03)00174-2 

Garner DL, Johnson LA, 1995. Viability assessment of mammalian sperm using SYBR-14 and propidium iodide. Biol Reprod 53: 276-284.
http://dx.doi.org/10.1095/biolreprod53.2.276
PMid:7492679  

Harrison RAP, Vickers SE, 1990. Use of fluorescent-probes to assess membrane integrity in mammalian spermatozoa. J Reprod Fertil 88: 343-352.
http://dx.doi.org/10.1530/jrf.0.0880343
PMid:1690300  

Nagy S, Jansen J, Topper EK, Gadella BM, 2003. A triple-stain flow cytometric method to assess plasma- and acrosome-membrane integrity of cryopreserved bovine sperm immediately after thawing in presence of egg-yolk particles. Biol Reprod 68: 1828-1835.
http://dx.doi.org/10.1095/biolreprod.102.011445
PMid:12606354  

Pintado B, De La Fuente J, Roldan ERS, 2000. Permeability of boar and bull spermatozoa to the nucleic acid stains propidium iodide or Hoechst 33258, or to eosin: accuracy in the assessment of cell viability. J Reprod Fertil 118: 145-152.
PMid:10793636  

Yaniz JL, Santolaria P, Marco-Aguado MA, Lopez-Gatius F, 2008. Use of image analysis to assess the plasma membrane integrity of ram spermatozoa in different diluents. Theriogenology 70: 192-198.
http://dx.doi.org/10.1016/j.theriogenology.2008.03.002
PMid:18440060  

Yaniz JL, Mateos JA, Santolaria P, 2011. Zwitterionic buffers preserve ram semen quality more efficiently than Tris during storage at 15 degrees C. Small Rumin Res 95: 54-60.
http://dx.doi.org/10.1016/j.smallrumres.2010.08.006 

Yaniz JL, Mateos JA, Santolaria P, 2012. Tris buffer improves fluorescence yield of ram spermatozoa when evaluating membrane integrity. Microscop Res Techn 75: 520-523.
http://dx.doi.org/10.1002/jemt.21086
PMid:22553829




DOI: 10.5424/sjar/2013112-3494