Villaverde Morcillo, SilviaEsteso Dïez, Milagros CCastaño García, CristinaToledano Díaz, AdolfoLópez Sebastián, AntonioCampo, José LuisSantiago Moreno, Julián2025-01-232025-01-232015Villaverde-Morcillo, Esteso, Castaño, Toledano Díaz, López-Sebastián, Campo, & Santiago-Moreno. (2015). Influence of Staining Method on the Values of Avian Sperm Head Morphometric Variables. Reproduction in Domestic Animals, 50(5), 750-755. https://doi.org/10.1111/RDA.125740936-676810.1111/rda.12574https://hdl.handle.net/20.500.14352/115912Author contributions: S. Villaverde-Morcillo performed the semen recovery and analysis,analysed the data and writing the basis of the article; M.C. Estesoperformed conception and design of the study, discussed the data andapproved the final draft; C. Casta~no worked on animal management,semen collection and analysis and approved the final draft; A.Toledano D ıaz involved in management of animals and worked ondata recording during semen collection; A. L opez-Sebasti an discussedthe data and approved the final draft; J.L. Campo discussed the dataand approved the final draft; and J. Santiago-Moreno performedconception and design of the study, participation and generalsupervision of the collection of data, writing the basis of the articleand approvedComputer-assisted systems for the assessment of sperm morphometry (ASMA systems) have been used successfully with several mammalian species. Unfortunately, they have so far been of little use for assessing bird semen, a consequence of the filiform shape of avian spermatozoa. This study compares two staining techniques (Hemacolor(®) and aniline blue staining) for the morphometric analysis of rooster and red-legged partridge spermatozoa as part of a computer-assisted light microscopy method. For both species, Hemacolor(®) staining provided a significantly higher percentage of measurable cells (93.7 ± 11.7% in roosters and 71.9 ± 15.3% in red-legged partridges). Hemacolor(®) also showed greater repeatability (lower coefficients of variation) for length and area in roosters' sperm and for width in the case of red-legged partridge's sperm. In the roosters, the Hemacolor(®) technique returned significantly (p < 0.05) larger sperm head width and area values than did the aniline blue technique, while the latter resulted in greater sperm head length values (p < 0.05). In the red-legged partridge, no differences were seen in the results for sperm head width and area provided by the two techniques, but aniline blue staining was associated with longer length measurements. In conclusion, the morphometric values recorded differed depending on the staining method and species. However, the Hemacolor(®) technique might be deemed the more appropriate for computerized sperm assessment systems as it provides larger percentages of measureable cells and shows greater repeatability.engjournal article1439-0531https://doi.org/10.1111/rda.1257426192019restricted access636.082.4Avian spermatozoaSperm morphometryHemacolorAnilineCiencias31 Ciencias Agrarias