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.
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.
30. Yaniz J.L., Soler C., Santolaria P.: Computer assisted sperm morphometry in mammals: a review. Anim Reprod Sci 2015, 156, 1-12.