Adenosine supplementation enhances chilled semen quality in Assaf rams: dose-dependent effects on sperm motility, membrane integrity, and morphology.

Ram semen is sensitive to thermal and oxidative stress due to greater concentrations of polyunsaturated fatty acids (PUFA) in the sperm cell membrane, rendering it inherently fragile during preservation. Adenosine (9-β-D-Ribofuranosyladenine) serves as an essential component in adenosine monophosphate (AMP) and adenosine triphosphate (ATP) synthesis, and plays a key role in sperm energy metabolism and function. This study investigated the effects of adding different adenosine concentrations (0%, 0.5%, 0.75%, and 1%) to Tris-egg yolk extender on the quality of chilled semen of Assaf rams. Semen was collected from seven rams every five days over a one-month period at 7:30 AM, pooled, and extended in Tris-egg yolk, after which adenosine stock solution was added at the designated concentrations. Samples were then cooled to 4°C and stored for 72 h. Motility, viability, membrane integrity, and morphology were assessed at 0, 24, 48, and 72 h. The data were subjected to analysis of variance and comparison of means using Bonferroni test. Incorporating 0.75% adenosine significantly improved semen quality at 48 and 72 h versus other groups (P < 0.05 for all parameters). At 48 and 72 h, respectively, the 0.75% group showed higher total motility (70.9% vs. 60.1%; 56.7% vs. 44.4%), progressive motility (45.2% vs. 34.8%; 39.4% vs. 25.8%), membrane integrity (HOST-positive: 70.3% vs. 64.2%; 68.2% vs. 55.3%), and viability (68.5% vs. 61.2%; 61.8% vs. 52.1%) than the control, with fewer abnormal spermatozoa (12.7% vs. 18.4%; 14.8% vs. 23.5%). Storage time also significantly affected all parameters (P < 0.05), with no significant adenosine × time interaction (P > 0.05). These findings have practical implications for improving sheep reproductive efficiency, given the physiological fragility of the PUFA-rich ovine sperm membrane. Supplementing Tris-egg yolk with 0.75% adenosine is recommended to extend the usable storage life of chilled ram semen up to 72 h, offering greater flexibility in the scheduling and logistics of artificial insemination programs, pending further in vivo fertility trials and comparisons with commercial extenders. Whether this beneficial effect extends to ram semen cryopreservation remains an open question for future research.