In contrast to the livestock industry, sperm cryopreservation has not yet been successfully established in the poultry industry. This is because poultry sperm cells have a unique shape and membrane fluidity, differing from those of livestock sperm. The objective of this review is to discuss the cellular and molecular characteristics of rooster spermatozoa as a cause for their generally low freezability. Furthermore, here, we discuss novel developments in the field of semen extenders, cryoprotectants, and freezing processes, all with the purpose of increasing the potential of rooster sperm cryopreservation. Currently, it is very important to improve cryopreservation of rooster sperm on a global scale for the protection of gene resources due to the incidence of epidemics such as avian influenza.

Spermatozoa cryoconservation represents an important strategy for partial in vitro or rescue programs designed for threatened livestock populations. The procedure for the semen cryopreservation of the Czech Golden Spotted Hen was proposed due to the lower fertilization rate of poultry semen compared to mammalian species. The aim of this study was to compare commercial extenders designed for liquid storage preservation with the use of a predefined cryoprotectant, and, thus, to propose an important tool for the procedure of the semen cryopreservation of the Czech Golden Spotted Hen. Ejaculates were sampled from four roosters during five semen collection days. The samples were frozen in Poultry media^®, Raptac^® and NeXcell^® extenders supplemented with a 9% N-methylacetamide (NMA) cryoprotectant. Sperm parameters of the total motility (MOT; %), plasma membrane and acrosome intactness (PAI; %), plasma membrane damage (%), acrosome damage (%) and cells with plasma membrane and acrosome damage (%) were assessed using a mobile mCASA analyzer and flow cytometer after the cryopreservation of the insemination doses (IDs). For Poultry media^® (PAI = 51.11%; MOT = 23.58%) and Raptac^® (PAI = 52.04%; MOT = 23.13%) extenders with the addition of an NMA cryoprotectant, the comparable results were detected after thawing. For NexCell^® media, the results were poor (PAI = 7.07%; MOT = 3.83%). Our results indicated two extenders suitable for the cryopreservation procedure, with the applied modification.

The objective of this study was to evaluate the impact of the supplementation of varying concentrations of the impermeable disaccharide trehalose on the in vitro and in vivo fertilization capacity of cryopreserved rooster spermatozoa in the original Czech Golden Spotted Hen breed. The control trehalose concentration was 0 mM, while TRE50 (50 mM), TRE100 (100 mM), and TRE200 (200 mM) were used as experimental trehalose concentrations. The kinematic and functional parameters of frozen/thawed spermatozoa were evaluated in vitro using mobile computer-assisted sperm analysis and a flow cytometer. The addition of 100 mM trehalose demonstrated the most favorable results for total (34.17%) and progressive (3.57%) motility after thawing. A statistically significant difference was found for these kinetic parameters compared to the other monitored concentrations. This experimental group was also found to have a significantly higher percentage of spermatozoa without plasma membrane or acrosome damage (33.37%) compared to the TRE50 group (30.74%; p < 0.05) and the TRE200 group (29.05%; p < 0.05). In vivo, artificial insemination was performed to verify fertilization ability. Hens (n = 40) were artificially inseminated twice (10 hens/treatment) with a 3-day interval between inseminations. In conclusion, the addition of 100 mM trehalose significantly improved total and progressive motility after thawing and preserved plasma membrane and acrosome integrity (p < 0.05). The fertilization rate of eggs fertilized with semen frozen with the addition of 100 mM trehalose was not significantly different from the other concentrations tested or the control group but was numerically higher (23.21% vs. 15.20% of fertilized eggs in this group).