The Influence of Three Commercial Soy Lecithin-Based Semen Extenders and Two Spermatozoa Concentrations on the Quality of Pre-Freeze and Post-Thaw Ram Epididymal Spermatozoa

Simple Summary: Studies have revealed a rapid global and continental loss of genetic resources for native sheep breeds that is more critical in Europe and the Caucasus region. Therefore, an urgent step is needed to halt this negative trend. Viable and functional epididymal spermatozoa could be retrieved from castrated, slaughtered, or accidentally dead animals with good pregnancy outcomes; however, many factors reported to affect the quality and cryo-tolerance of artificial vagina-collected, as well as electro-ejaculated, ram spermatozoa have not been extensively studied in ram epididymal spermatozoa despite being a cheap alternative for gene conservation. Given the context mentioned above, we assessed the effects of three different commercial soy lecithin-based semen extenders (AndroMed (R), BioXcell (R), and OviXcell (R)) and two spermatozoa concentrations (200 x 10(6)/mL vs. 400 x 10(6)/mL) on the freezability of ram epididymal spermatozoa. BioXcell (R) and OviXcell (R) produced significantly higher post-thaw specific kinematics and better protected the ram epididymal spermatozoa head membrane compared to AndroMed (R). In contrast, the normal tail morphology is better maintained in AndroMed (R). The 400 x 10(6) spermatozoa/mL concentration better preserved the ram epididymal spermatozoa head membrane integrity. The ideal concentration for cryopreserving ram epididymal spermatozoa is 400 x 10(6) spermatozoa/mL. However, the extenders must be optimized for more effective ram epididymal spermatozoa freezing. There are limited studies on the factors affecting the success of ram epididymal spermatozoa (REPS) cryopreservation. On this note, the current study assessed the influence of three commercial soy lecithin-based semen extenders, AndroMed (R) (AND), BioXcell (R) (BIO), and OviXcell (R) (OVI), and two concentrations (400 x 10(6) vs. 200 x 10(6) spermatozoa/mL) on the pre-freeze and post-thaw quality of REPS. The REPS were retrieved from nine adult rams' testes and diluted with each of the three extenders to both concentrations. Straws were frozen manually. Standard motility (SMP) and kinematic parameters (KPs) were assessed via a CASA, while spermatozoa viability, morphology, and acrosomal integrity were assessed via the Kov & aacute;cs-Foote staining technique. The concentration did not significantly affect the pre-freeze and post-thaw SMP and KPs of REPS. BIO and OVI had significantly higher pre-freeze and post-thaw BCFs, post-thaw VAP, and the percentage of all intact heads than AND. In contrast, AND had a significantly lower percentage of REPS with tail defects than BIO and OVI. The 400 x 10(6) spermatozoa/mL concentration resulted in a significantly higher percentage of all intact heads than the 200 x 10(6) spermatozoa/mL concentration. Freezing significantly increased tail defects and decreased the percentage of REPS with distal cytoplasmic droplets. The cryopreservation of REPS at the 400 x 10(6) spermatozoa/mL concentration is recommended. All three extenders must be optimized to preserve the viability, membrane integrity, and better normal morphology of REPS; the reason for increased tail abnormality after the freezing/thawing process needs to be studied.