Identifying a suitable semen cryopreservation protocol for the restricted ovulatory leghorn chicken by screening permeable and non-permeable cryoprotectant agents

Since its discovery in the early 1970's, there has been a substantial amount of research published on the restricted ovulatory (RO) leghorn chicken research line. This line possesses a paternally inherited mutation in the very-low density lipoprotein receptor, which causes follicles in the ovary not to fully develop, sterilizing the RO hens. Although well studied, this line still does not have a suitable means of semen cryopreservation and instead relies solely on living populations. Identifying a suitable semen cryopreservation protocol would ensure the availability of the RO line for future poultry researchers. For this study, semen was treated with permeable and non-permeable cryoprotectant agents (CPAs) or a combination, at varying concentrations. Permeable CPAs tested were dimethylformamide (DMF), dimethylacetamide (DMA), dimethyl sulfoxide (DMSO), and ethylene glycol (EG), along with two non-permeable CPAs, sucrose and trehalose. Motility parameters, membrane integrity, fertility, and hatchability were evaluated after semen thawing. For the RO semen, DMF 8% + trehalose 50 mM had one of the highest total motility (53.2 ± 3.3%), progressive motility (29.1 ± 4.8%), and membrane integrity (59.6 ± 2.2%) values. This protocol also had higher (P < 0.001) candled fertility (57%) during the first week after inseminations compared to DMF 8%, and DMF 8% + EG 2%. There was also no difference between this treatment's hatchability (92%) and the fresh-extended control. RO semen slow-frozen using DMF 8% + trehalose 50 mM gave high enough fertility and hatchability to be able to use for biobanking purposes.