North African houbara semen cryopreservation

Sperm cryopreservation is a strategy for long term ex-situ conservation of wild species, guarantees the continuity of breeding and supports long-term maintenance of genetic management. In North African Houbara Bustard (Chlamydotis undulata undulata) conservation breeding, reproduction is based on artificial insemination with fresh, refrigerated, and frozen doses. From 2000 to 2021, 21 960 ejaculates were frozen, and 3 500 inseminations performed with thawed sperm. Thus, the quality of the frozen samples is important for the successful use of this biotechnology. The aim of this study was to evaluate the impacts of cryopreservation on Houbara sperm quality. Semen was collected from 34 adult males (4 to 6y old), one aliquot was reserved for fresh sperm analysis (20µL in 180µL Lake 7.1, 1.5% BSA), the remaining diluted in Lake 7.1 and refrigerated for 10min/5℃, 6% DMA was added and frozen by pellet method. After 10 d, samples were thawed at 65℃ in a conic plate, a 20µL aliquot diluted in 180µL Lake 7.1, 1.5% BSA. Fresh and frozen-thawed samples were analysed for motility by computer assisted sperm analysis (CASA-SCA), membrane integrity (PI, Sybr 14), DNA compaction (HDS) and DNA strand breaks (%DFI). A paired Student’s t-test and Wilcoxon signed-rank test were used to compare sperm quality between groups. Motility and viability decreased significantly after cryopreservation (motile - 81.20%, curvilinear velocity -40.22%, straight-line velocity -64.03%, viability -8.33%, P0.05); however, HDS% was 78.74% higher on frozen/thawed samples. Whereas motility, viability, and sperm DNA compaction (HDS) were impaired by cryopreservation, %DFI was not affected. Avian sperm DNA compaction, but also DNA organization, need further investigations, particularly the key role of avian protamine.