Erythropoietin sustains functional and biochemical integrity of ram spermatozoa during prolonged chilled storage

Erythropoietin (EPO) is primarily known for its role in erythropoiesis. However, recent data confirm the presence and function (autocrine or paracrine) of EPO in human and rat testicular tissue and seminal plasma, suggesting a role in reproductive physiology. Moreover, a cytoprotective and antioxidant role of EPO has been documented in thawed bovine semen. Based on these, the present study aims to explore the possible effect of EPO on various parameters of fresh ram spermatozoa during prolonged cold storage. Semen from five Chios rams was supplemented with EPO at two concentrations [10 (EPO 10) and 100 mIU/μl (EPO 100)] or left untreated (control) and stored at 5°C for up to 3 days. Sperm motility, viability, oxidative status, apoptotic markers, and heat shock response were evaluated. EPO, and more specifically the 100 mIU/μl concentration, significantly mitigated the decline in motility parameters from Day 1 to Day 3 compared to the control. Computer Assisted Sperm Analyzer (CASA) parameters and glutathione (GSH) levels were better preserved, indicating maintenance of flagellar activity, membrane integrity, and antioxidant capacity. In addition, apoptotic signaling, as witnessed by a Bax/Bcl-2 ratio decrease, and Heat Shock Protein (Hsp70) expression were modulated by EPO, consistent with the potential involvement of cytoprotective pathways. These findings offer new insights into the role of EPO in the field of reproductive physiology, indicating that EPO helps ram spermatozoa to maintain their homeostasis.