Cryopreserved Sperm Quality in Tsigai Sheep: Implications for Biodiversity Protection

The objective of our research was to conduct a comparative analysis of cryopreserved sperm quality parameters of Tsigai (TS) sheep breed. Ejaculates (n=12) from TS sheep rams (n = 2) were collected using electro-ejaculation. Samples were prepared from suitable ejaculates, ensuring at least 70% progressive motility. These samples were then equilibrated in Triladyl® diluent and subjected to automated freezing. Before freezing and after thawing, the sperm samples underwent assessments including motility (CASA), viability and apoptosis (DRAQ7/Yo-Pro-1), mitochondrial activity (MitoTracker), capacitation status (FLUO4), and acrosomal status (PNA). The results revealed significant differences (P < 0.05) in total motility between fresh (91.25±0.80 %) and frozen/thawed (F/T) (41.14±12.04 %) samples, as well as progressive motility between fresh (89.83±1.85 %) and frozen/thawed (33.57±8.25 %) groups. Moreover, significant differences (P<0.05) were also observed in the proportion of apoptotic spermatozoa (2.26±0.60 %), dead (7.40±1.72 %), and mitochondrial active spermatozoa (84.52±5.60 %) in fresh samples compared to F/T samples (12.34±0.80 %, 46.59±2.82 %, 47.71±4.04 %). Our findings indicate that the selected cryopreservation protocol is relatively sufficient for the cryoconservation of sperm from Tsigai sheep breeds, since up to 50% of F/T sperm were motile and live. This has significant implications for biodiversity protection and simplifies the establishment of an animal genetic resources gene bank.