Comparison of assessment of pigeon sperm viability by contrast-phase microscope (eosin-nigrosin staining) and flow cytometry (SYBR-14/propidium iodide (PI) staining) [evaluation of pigeon sperm viability

Abstract

The aim of these experiments was to compare the conventional, microscopic method of evaluating pigeon sperm viability to sperm assessed by flow cytometry. Semen was collected twice a week from two groups of pigeons. In every group were 20 males (Group I: meat-type breed; Group II: fancy pigeon breed). Semen was collected using the lumbosacral and cloacal region massage method. Ejaculates collected from each group were pooled and diluted to 10 × 10 6 sperm/ml in BPSE solution. Samples were divided into three equal parts and estimated after collection as well as after in vitro storage for 3, 6 and 24 h. The first part was using for semen motility evaluation. The proportion of motile spermatozoa (MOT) and progressive movement (PMOT) of fresh and stored semen were evaluated using the CASA-system. The second part was examined subjectively by microscope (eosin-nigrosin (EN), eosin-nigrosin staining), the third one was assessed using dual fluorescence SYBR-14/propidium iodide (PI) and flow cytometry (FC). There were not any significant differences in sperm viability and motility between the groups at 0, 3, 6, and 24 h post collection. The percentage of viable spermatozoa in fresh semen determined by EN and FC was not different in Groups I and II (I - 88.71 ± 5.42 and 84.01 ± 3.19, respectively; II-90.87 ± 6.01 and 87.38 ± 5.57, respectively). Significantly lower percentages of viable spermatozoa were detected by FC compared to the EN method in both groups after 6 h (P ≤ 0.05) as well as 24 h (P ≤ 0.01) of storage. Moreover, the dual fluorescent SYBR-14/PI staining allowed for the identification a third population of double stained, moribund spermatozoa. High positive correlations in percentage of live spermatozoa were noted between EN and FC methods in both groups of birds. Evaluation of sperm viability by FC is a rapid, accurate, sensitive, and objective method for the assessment of pigeon sperm viability in fresh as well as stored semen.