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Methodological variants contributing to detection of abnormal DNA-protein complexes in bull spermatozoa^*

Marcelo Emilio Beletti^I,II; Maria Luiza S, Mello^I

^IDepartamento de Biologia Celular, Instituto de Biologia, UNICAMP, 13083-970 Campinas, SP, Brasil
^IIDepartamento de Biologia, Faculdade de Veterinária de Pinhal, FPE, 13990-000 Espírito Santo do Pinhal, SP, Brasil. Send correspondence to M.E.B.

ABSTRACT

Efficient, simple and inexpensive methods for detecting abnormal DNA-protein complexes in sperm cells present in the semen of bulls with different fertility characteristics were tested cytochemically. The methods are based on nuclear metachromasy induced after toluidine blue staining and on different fluorescence emissions after acridine orange staining of spermatozoal nuclei bearing abnormal DNA-protein complexes. Treatments with 2-mercaptoethanol plus urea and NaCl, with SSC solution and with 4 N HCl were found to be the most efficient for inducing nuclear metachromasy. The 4 N HCl treatment was found to be the least efficient among these, though the simplest and cheapest. With the methods employing acridine orange as the dye solution, the frequency of sperm cell nuclei exhibiting high fluorescence intensity resembled that of the metachromatic nuclei highlighted with toluidine blue staining, if preceded by treatment with 4 N HCl or 2% citric acid. Although the animals with morphological defects associated with low fertility rates generally exhibited a higher frequency of metachromatic sperm cell nuclei and greater acridine orange (AO) fluorescence intensity, this could not be taken as a precise rule.

Keywords: DNA-protein; bull spermatozoa.

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* Part of a thesis presented by M.E.B. to the Instituto de Biologia, Universidade Estadual de Campinas (UNICAMP), Campinas, SP, in partial fulfillment of the requirements for the Master's degree.