Impact of semen extenders, storage duration, and insemination timing on semen quality and reproductive performance in Palestinian Assaf sheep

Background and Aim: Artificial insemination (AI) is a critical technique in sheep breeding programs, yet its success is influenced by factors such as semen quality, storage conditions, and insemination timing. This study examines the effects of different semen extenders, storage durations, and insemination schedules on semen motility and pregnancy rates in Palestinian Assaf sheep. The primary aim of this study was to assess and compare the efficacy of three semen extenders – Andromed, Indonesian, and Syrian – on the quality of stored semen and its subsequent effect on pregnancy rates following AI. In addition, the study investigated the impact of semen storage duration and the timing of insemination post-equine chorionic gonadotropin (eCG) injection on reproductive outcomes. Materials and Methods: Semen was collected from eight Assaf rams, diluted with one of three extenders, and stored at 4°C for up to 72 h. Sperm motility was analyzed using a computer-assisted sperm analysis system at different time intervals. AI was performed on 180 Assaf ewes across three farms in Palestine, with insemination conducted at either 48 h, 52 h, or both 48 and 52 h post-eCG injection. Pregnancy rates were determined through ultrasound 45 days post-insemination. Statistical analysis was conducted using the Statistical Package for the Social Sciences 22 (IBM® NY, USA), with p-values set at <0.05 for significance. Results: Semen motility decreased significantly over time, with total motility (TM) declining from 0.85 ± 0.01 (fresh) to 0.63 ± 0.02 after 3 days of storage. The Syrian extender exhibited superior fast motility (FM) (0.35 ± 0.01) compared to the others. Strong positive correlations were observed between TM and progressive motility (0.90, p ≤ 0.01). Pregnancy rates did not significantly differ among extenders (Andromed: 0.58 ± 0.06, Indonesian: 0.54 ± 0.07, Syrian: 0.56 ± 0.08). However, insemination performed at both 48 and 52 h post-eCG injection resulted in the highest pregnancy rate (0.62 ± 0.07), while the second ejaculation showed a tendency for improved fertility outcomes (0.61 ± 0.07). Conclusion: Prolonged semen storage negatively impacts motility, though the Syrian extender preserves FM better than the others. Pregnancy rates were not significantly influenced by the extender type but were optimized by insemination at both 48 and 52 h post-eCG injection. These findings highlight the importance of refining semen preservation techniques and timing AI procedures to enhance breeding success in Assaf sheep.