Effect of initial ejaculate quality on post-thaw parameters in boar semen

The initial ejaculate sperm quality influences its subsequent freezability, which varies significantly between individuals. The objective of this study was to determine whether non-viable, static, or morphologically altered sperm affect the freezability of motile, viable sperm with normal morphology. Semen samples were collected from five sexually mature boars (≥3 ejaculates per animal) and allocated to four treatment groups based on the proportion of viable, motile, morphologically normal sperm: 100% (control), 75%, 50%, and 25%. Samples were diluted in TRIS-egg yolk extender, cooled to 5 °C, then further diluted with a glycerol-containing freezing medium and loaded to 0.5-ml straws. The cryopreservation protocol involved two controlled cooling stages, followed by storage in liquid nitrogen for seven days. After thawing at 37 °C for 20 s, sperm quality was evaluated using computer-assisted semen analysis (CASA) for motility and flow cytometry for membrane and acrosome integrity with triple fluorescent staining (Hoechst 33342, propidium iodide, and PNA-FITC). Assessments were carried out before freezing and at 30 and 150 min post-thaw. The percentage of non-viable sperm in the ejaculates prior to freezing significantly affected post-thaw sperm quality (P < 0.05), reducing motility and structural integrity in proportion to their initial presence.