Exploration of Semen Cryopreservation Protocol for Horasi Chicken Ecotype

To establish the most effective cryopreservation protocol for the Horasi chicken ecotype semen, sperm traits, such as viability, motility, and morphology, were examined under various settings using different extenders (Beltsville Poultry Semen Extender (BPSE), LAKE, and CARI), cryoprotectants (dimethyl sulfoxide (DMSO), dimethyl acetamide (DMA), and dimethyl formamide (DMF)), concentrations for each cryoprotectant (4%, 8%, and 12%), equilibration conditions (4°C for 30 min, 4°C for 2 h, and 4°C for 3 h), and thawing conditions (5°C for 100 s, 35°C for 60 s, and 37°C for 30 s). Between December 12, 2024, and March 19, 2025, a total of 243 protocols with three replicates each, making a total of 729 semen samples, were tested. Semen samples were obtained from 10 Horasi cockerels by employing an abdomen massaging technique, evaluated, pooled, processed, and cryopreserved in liquid nitrogen gas (−196°C) for 24 h. The highest values for total motility (92.68 ± 2.46), progressive motility (70.31 ± 1.24), viability (91.67 ± 2.88), and sperm with normal morphology (88.33 ± 2.88) were obtained in semen samples extended with BPSE and 4% DMSO, equilibrated at 4°C for 2 h, and thawed at 5°C for 100 s, whereas the lowest number of motile (1.56 ± 1.25), progressive motile (0.10 ± 0.58), viable (16.33 ± 1.30), and morphologically normal (9.33 ± 1.15) sperm cells were recorded in semen diluted with LAKE extender containing 12% DMA, equilibrated at 4°C for 3 h, and thawed at 37°C for 30 s. In summary, our findings demonstrate the critical stages of the procedure and the ideal conditions, marking a significant advancement in the research on the Horasi chicken ecotype semen cryopreservation technique.