Constitution of Nero Siciliano pig breed genetic reserve: flow cytometry evaluation of epididymal sperm cells

In the framework of the research program "Integrate knowledge for sustainability and innovation of the agro-food Made in Italy (CISIA)" the genetic reserve of the Nero Siciliano local pig breed was created. This breed is farmed in marginal areas and few males are available for semen collection. In this context epididymal sperm extraction was the only option to collect gametes. Semen quality evaluation and determination of its fertilizing ability are essential aspects in the practice of assisted reproduction in domestic species, especially in local breeds, where few chances to store semen are available. With flow cytometry (FCM) technique an objective and quick assessment of semen potential fertility is obtainable. The aim of this study was to evaluate different quality parameters of boar frozen-thawed epididymal sperm by using advanced technologies as FCM and computer assisted sperm analysis (CASA). Testicles were obtained from 12 boars after surgical castration performed directly on farm. Epididymal sperm were extracted from cauda epididymis by using the retrograde flushing technique. At freezing-thawing, the following sperm parameters were evaluated by FCM: viability (SYBR-14/propidium iodide (PI) staining), acrosomal status (FITC-PNA/PI staining), membrane lipid disorder (Merocyanine 540 hydrophobic dye), mitochondrial membrane potential (JC-1 potentiometric probe) and sperm DNA fragmentation (acridine orange dye). Sperm motility and kinetic parameters were assessed by CASA system. A total of 1401 semen doses of Nero Siciliano local pig breed were stored with suitable sperm quality. The mean values of measured parameters, standard deviations, minimum and maximum values, on frozen-thawed samples, were: viable sperm: 41.3±9.2 % (31.0-61.8); live acrosome intact sperm: 34.6±9.7 % (18.9-52.9); live sperm with low lipid disorder: 26.6±9.5% (15.6-48.3); sperm with high mitopotential: 38.7±10.6% (22.1-61.2); sperm DNA fragmentation index (% DFI): 5.6±2.7 (2.6-11.6); total motile sperm: 44.0 ± 12.0% (21.2-68.0); curvilinear velocity: 89.9±9.2 µm/s (77.3-103.8); average path velocity: 39.1±7.7 µm/s (25.0-47.8); straight-line velocity: 8.4±2.8 µm/s (5.5-14.0); linearity index: 9.2±2.7% (6.5-14.2). The current study is a concrete example of the application of advanced techniques, like FCM or CASA, for the validation of frozen-thawed epididymal boar sperm as gametes source in animal genetic resources cryobanks.