Comprehensive Statistical Assessment of Post-Thaw Semen Quality in Two Conserved Norwegian Rooster Lines

The conservation of local poultry genetic resources requires complementary ex situ strategies, particularly when live populations are maintained at a single location. Previous gene bank insemination trials with frozen-thawed semen from Norwegian conserved lines showed acceptable subjective post-thaw motility but unexpectedly low fertilization success, highlighting the need for objective quality-control assessment. The aim of this study was to establish an objective cryobank quality-control baseline for frozen-thawed semen from two Norwegian conserved rooster lines. Specifically, the study evaluated whether routinely applied objective semen-quality assessment methods, including computer-assisted semen analysis (CASA) and flow-cytometric viability assessment, could detect differences between rooster lines and post-thaw handling conditions. Cryopreserved semen doses collected in 2018 and stored in the Norwegian poultry gene bank were thawed and evaluated from 24 roosters representing the Jærhøns and NorBrid8 lines. The samples were originally collected as part of the routine cryopreservation activities of the Norwegian poultry gene bank and therefore did not require specific approval from the Norwegian Food Safety Authority (Mattilsynet). Post-thaw semen quality was assessed using CASA and flow-cytometric viability analysis. The effects of line and short-term refrigerated holding were evaluated using mixed-effects models, supported by minimum detectable effect calculations, correlation analysis, and principal component analysis. Short-term post-thaw refrigerated holding reduced several CASA-derived motility and velocity traits, while viability assessment provided complementary information on membrane integrity. Line-associated differences in selected motility traits and substantial rooster-to-rooster variability in all traits were observed. Multivariate analyses identified major dimensions of covariance among sperm-quality traits without implying direct biological mechanisms of fertility failure. CASA and flow-cytometric viability assessment can provide an objective baseline for cryobank quality control of frozen-thawed rooster semen. However, fertility trials and molecular sperm-integrity markers are required to determine the biological causes of the previously observed discrepancy between motility and fertility.