The aim of this experiment was to study the effect of semen extender on sperm chromatin structure and to correlate chromatin integrity with field-fertility of preserved ram semen. Ejaculates of at least 2 × 109 sperm/ml and 70 % progressive motility were collected using an artificial vagina from Chios rams (n = 11, 4–6 years old), split-diluted to 1 × 109 sperm/ml with milk-egg yolk- and soybean lecithin (Ovixcell®)-based extenders, packaged in 0.5-ml straws and examined after 6, 24 and 48 h of storage at 5 ± 1 °C. Evaluation endpoints were computer-assisted sperm motion analysis, fluorescence-based analysis of chromatin structure by chromomycin A3 and acridine orange assays, and 65-day pregnancy rate (PR) of 34- to 36-h preserved semen after intra-cervical insemination of ewes (n = 154) in progestagen-synchronized estrus. Neither extender nor storage time had any influence on incidence of decondensed chromatin. Unlike Ovixcell® extender, deterioration of sperm motility (P < 0.01) and chromatin stability (P < 0.005) was detected after 48 h of storage in milk-egg yolk extender. Sperm motility accounted for 14.4–18.5 % of variations in chromatin integrity (P < 0.001). No significant difference was found in PR of Ovixcell®- and milk-egg yolk-stored semen. Nevertheless, PR differed between rams (14.3–71.4 %; P < 0.025). Chromatin integrity explained 10.2–56.3 % of variations in PR (P < 0.05–0.01). A pronounced decline in PR (19.1 %) was observed when percentages of decondensed and destabilized chromatin have reached thresholds of 10.5–30 % and 4–9 %, respectively. In conclusion, Ovixcell® is superior to milk-egg yolk extender in preserving chromatin stability and motility. Chromatin defects are negatively associated with sperm fertility.
