Influence of Staining Method on the Values of Avian Sperm Head Morphometric Variables

Abstract

Computer-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.

Recent advances in bird sperm morphometric analysis and its role in male gamete characterization and reproduction technologies

“…For example, the choice of fixative (e.g., methanol, glutaraldehyde) employed when stains are used is known to modify the size of avian spermatozoa 5. The osmotic stress resulting from the use of hyper- or hypo-tonic solutions to dilute semen may also affect sperm size via the dehydration or swelling of cells.…”

“…Indeed, shrinkage often occurs when biological material is prepared for electron microscopy 58. This may explain why both chicken and turkey sperm morphometric variables are usually recorded as smaller when examined in the electron microscope5354 than in the light microscope 557…”

“…Abnormal spermatids are destroyed by the Sertoli cells via induced apoptosis, followed by the engulfing and digestion of the debris. In turkeys suffering from yellow semen syndrome, abnormal spermatids appear in the semen;4 in partridges and roosters, spermatids are not seen in normal ejaculates,5 but in falcons, nonapoptotic spherical spermatids (immature spermatids) are habitually seen in the lumen of the seminiferous tubule and in normal ejaculates, although their physiological role in reproduction is unknown.…”

Current status and potential of morphometric sperm analysis

“…All these steps can affect not only the repeatability of the experiments but also the reproducibility and the comparison of results among laboratories, which are necessary for the practical use of sperm morphometric analyses. These aspects have been studied by several authors,6465666768697071 and they have not yet been completely resolved.…”

“…The choice of staining protocol is the issue on which most authors have conducted their studies63656668707172737980 since it influences background noise, sperm contrast, and the identification of different areas within the cell. To prevent these problems during morphometric sperm analysis, fluorescence-based methods in combination with a CASA-Morph system have been developed for a more precise measurement of the nucleus, acrosome, and sperm head, yielding promising results 6481.…”