Introduction: The effect of two smear staining methods on the dimensions and shape of sperm cells in the semen of domestic pigs was evaluated. Material and Methods: The studies were carried out on 30 ejaculates collected from 15 boars, which included five Duroc boars, five Pietrain boars, and five hybrid Duroc × Pietrain boars. Each ejaculate was next sampled to make two microscopic slides, of which one was stained with eosin-nigrosin and the other with eosin-gentian dye. In total, 600 measurements of sperm cells were made. Each sperm was measured for the following morphometric parameters: head length, head width, head area, head perimeter, tail length, and the total sperm length. Results: Sperms measured on slides stained with eosin-nigrosin showed lower dimensions as compared with those stained with the eosin-gentian dye method. Sperm stained with eosin-nigrosin had shorter and narrower heads than sperm stained with eosin-gentian dye. The method of staining, therefore, affected not only the dimensions of the sperm, but also the proportions of the dimensions defining the shape of the sperm. Conclusions: The size and shape parameters in porcine sperm may take on different values depending on the method of semen staining. Sperm cells stained with eosin-nigrosin are smaller than the sperm stained with eosin-gentian dye. The sensitivity of the sperm to the type of dye used for the fixation may be associated with genetic factors.
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1. Almadaly E., Farrag F., Shukry M., Murase T.: Plasma membrane integrity and morphology of frozen-thawed bull spermatozoa supplemented with desalted and lyophilized seminal plasma. Global Vet 2014, 13, 753-766.
2. Álvarez M., Garcia-Macias V., Martinez-Pastor F., Martinez F., Borragán S.: Effects of cryopreservation on head morphometry and its relation with chromatin status in brown bear (Ursus arctos) spermatozoa. Theriogenology 2008, 70, 1498-1506.
3. Andrabi S.M.H.: Mammalian sperm chromatin structure and assessment of DNA fragmentation. J Assist Reprod Genet 2007, 24, 561-569.
4. Björndahl L., SöderlundI., Johansson S., Mohammadieh M., Pourian M.R., Kvist U.: Why the WHO recommendations for eosin-nigrosin staining techniques for human sperm vitality assessment must change. J Androl 2004, 25, 671-678.
5. Brito L.F.C., Greene L.M., Kelleman A., Knobbe M., Turner R.: Effect of method and clinical on stallion sperm morphology evaluation. Theriogenology 2011, 76, 745-750.
6. Cardoso R.C.S., Silva A.R., Silva L.D.M.: Métodos de avaliacão do semen canino congelado. Rev Bras Reprod Anim 2005, 29, 179-187.
7. Enciso M., Cisale H., Johnson S.D., Sarasa J., Fernandez J.L., Gosalvez J.: Major morphological sperm abnormalities in the bull are related to sperm DNA damage. Theriogenology 2011, 76, 23-32.
8. Frenau G.E., Chenoweth P.J., Ellis R., Rupp G.: Sperm morphology of beef bulls evaluated by two different methods. Anim Reprod Sci 2010, 118, 176-181.
9. Gao D.Y., Ashworth E., Watson P.F., Kleinhans F.W., Mazur P., Crister J.K.: Hyperosmotic tolerance of human spermatozoa: separate effects of glycerol, sodium chloride, and sucrose on sperm analysis. Biol Reprod 1993, 49, 112-123.
10. Gil M.C., García-Herreros M., Barón F.J., Aparicio I.M., Santos A.J., García-Marín L.J.: Morphometry of porcine spermatozoa and its functional significance in relation with the motility parameters in fresh semen. Theriogenology 2009, 71, 254-263.
11. Katila T.: In vitro evaluation of frozen-thawed stallion semen: a review. Acta Vet Scand 2001, 42, 199-221.
12. Kondracki S., Iwanina M., Wysokińska A., Huszno M.: Comparative analysis of Duroc and Pietrain boar sperm morphology. Acta Vet Brno 2012, 81, 141-145.
13. Kowalewski D., Kondracki S., Górski K., Bajena M., Wysokińska A.: Effect of piggery microclimate on ejaculate performance of artificial insemination boars. Kafkas Univ Vet Fak Derg 2016, 22, 225-232.
14. Kruger T.F., Ackerman S.B., Simmons K.F., Swanson R.J., Brugo S.S., Acosta A.A.: A quick, reliable staining technique for human sperm morphology. Arch Androl 1987, 18, 275-277.
15. Kruger T.F., Lacquet F.A., Sarmiento C.A., Menkveld R., Ozgur K., Lombard C.J., Franken D.R.: A prospective study on the predictive value of normal sperm morphology as evaluated by computer (IVOS). Fertil Steril 1996, 66, 285-291.
16. Lavara R., Vincente J.S., Baselga M.: Genetic variation in head morphometry of rabbit sperm. Theriogenology 2013, 80, 313-318.
17. Łącka K., Kondracki S., Iwanina M., Wysokińska A.: Assessment of stallion semen morphology using two different staining methods, microscopic techniques, and sample sizes. J Vet Res 2016, 60, 99-104.
18. Łukaszewicz E., Jerysz A., Partyka A., Siudzińska A.: Efficacy of evaluation of rooster sperm morphology using different staining methods. Res Vet Sci 2008, 85, 583-588.
19. Maree L., du Plessis S.S., Menkveld R., van der Horst G.: Morphometric dimensions of the human sperm head depend on the staining method used. Hum Reprod 2010, 25, 1369-1382.
20. Maroto-Morales A., Ramón M., García-Álvarez O., Soler A.J., Esteso M.C., Martinez-Pastor F., Pérez-Guzmán M.D., Garde J.J.: Characterization of ram (Ovis aries) sperm head morphometry using the Sperm-Class Analyzer. Theriogenology 2010, 73, 437-448.
21. Oberlender G., Murgas L.D.S., Zangeronimo M.G., Silva A.C., Pereira L.J., Muzzi R.A.L.: Comparison of two different methods for evaluating boar semen morphology. Arch Med Vet 2012, 44, 201-205.
22. O’Connel M., McClure N., Lewis S.E.: The effect of cryopreservation on sperm morphology, motility, and mitochondrial function. Hum Reprod 2002, 17, 704-709.
23. Peña A.I., Lugilde L.L., Barrio M., Herradon P.G., Quintela L.A.: Effects of Equex from different sources on post-thaw survival, longevity and intracellular Ca2+ concentration of dog spermatozoa. Theriogenology 2003, 59, 1725-1739.
24. Phillips N.J., McGowan M.R., Johnston S.D., Mayer D.G.: Relationship between thirty post-thaw spermatozoal characteristics and the field fertility of 11 high-use Australian dairy AI sires. Anim Reprod Sci 2004, 81, 47-61.
25. Sancho M., Perez-Sanchez F., Tablado L., de Monserrat J.J., Soler C.: Computer assisted morphometric analysis of ram sperm heads: evaluation of different fixative techniques. Theriogenology 1998, 50, 27-37.
26. Thurston L.M., Watson P.F., Holt W.V.: Sources of variation in the morphological characteristics of sperm subpopulations assessed objectively by a novel automated sperm morphology analysis system. J Reprod Fertil 1999, 117, 271-280.
27. van der Horst G., Kitchin R.M., van der Horst M., Atherton R.W.: The effect of the breeding season, cryopreservation and physiological extender on selected sperm and semen parameters of four ferret species: implications for captive breeding in the endangered black-footed ferret. Reprod Fertil Dev 2009, 21, 351-363.
28. van der Horst G., Maree L.: SpermBlue: a new universal stain for human and animal sperm which is also amenable to automated sperm morphology analysis. Biotech Histochem 2009, 84, 299-308.
29. Wysokińska A., Kondracki S., Banaszewska D.: Morphometrical characteristics of spermatozoa in Polish Landrace Boars with regard to the number of spermatozoa in an ejaculate. Reprod Biol 2009, 9, 271-282.