55 COMPARATIVE STUDY BETWEEN PHASE CONTRAST AND DIFFERENTIAL INTERFERENCE CONTRAST MICROSCO PIES FOR EVALUATION OF FROZEN BULL SEMEN VALQUIRIA HYPPÓLITO BARNABE Professor Livre-Docente Faculdade de Medicina Veterinária e Zootecnia da USP RENATO CAMPANARUT BARNABE Professor Adjunto Faculdade de Medicina Veterinária e Zootecnia da USP JOSÉ ANTONIO VISINTIN Auxiliar de Ensino Faculdade de Medicina Veterinária e Zootecnia da USP WILSON GONÇALVES VIANA Professor Assistente Faculdade de Medicina Veterinária e Zootecnia da USP JOÃO FLORIANO CASAGRANDE Médico Veterinário SEMBRA - Técnicas e Produtos de Reprodução Ltda. - Barretos CARLOS ALBERTO DE ALMEIDA Médico Veterinário SEMBRA - Técnicas e Produtos de Reprodução Ltda. - Barretos BARNABE, V.H.; BARNABE, R.C.; VISINTIN, J.A.; VIANA, W. G.; CASAGRANDE, J.F.; ALMEIDA, C.A. Comparative study between phase contrast and differential interference contrast microscopies for evaluation of frozen bull semen. Rev.Fac. Med.vet.Zootec.Univ.S. Paulo, 18(1): 55-59,1981. 1981. SUMMARY: Two hundred ampules of frozen bull semen were eva- luated for per cent acrosomal pathology and major and minor de- fects of spermatozoa. The ampules referred to 4 groups of 50 each, corresponding to semen frozen in 1975, 1976, 1977 and 1978. Se- men examinations were made after thawing and after being placed in a 38° C water bath for 5 hours (Slow Thermoresistance Test) or in a 45° C water bath for 1 hour (Quick Thermoresistance Test). Analysis of variance showed highly significant differences (P < .01) between phase-contrast and differential interference contrast mi - croscopies for evaluation of acrosomal pathology and major defects of spermatozoa. For minor defccts analysis of variance did not show statistical differences between the two technics employed. UNITERMS: Differential interference contrast microscopy*; Phase- contrast microscopy* ; Frozen bull semen evaluation* Several studies have shown that impaired fertility in the bull may be related to morphologic defects in spermato- zoa. Thus, evaluation of spermatozoal morphologic features is an important aid in assessing a bull’s breeding soundness. Spermatozoal morphologic features have generally been evaluated in stained seminal smears. Although procedures for preparing stained smears may be detrimental to sperma- tozoa integrity5. Phase contrast and differential interference contrast microscopies make it possible to evaluate spermatozoa mor- phologic features in wet preparations of semen, with buffe- red formol saline or 0 .2% glutaraldehyde in phosphate-buf- fered saline. This last fixation procedure allows the trans- port of semen samples preventing cellular injuries9 and the possibility of storage up to 29 days6. Resolution of differential interference contrast is im- proved over that of phase contrast microscope because in- terference halos are greatly minimized10. Differential interference contrast microscope has been used for the study of correlations between spermatozoal abnormalities and fertility 10 and for routine evaluation of semen, including all the ejaculations of bulls in service1’2> 4,7,11. The objective in the present investigation was to com- pare phase contrast and differential interference contrast methods, regarding acrosome evaluation and major and mi- nor defects 3 in frozen semen of bulls. MATERIAL AND METHOD There were studied 200 ampules of frozen semen from 10 bulls, donors in an Artificial Insemination Centre placed in Barretos, São Paulo State, Brazil. The ampules referred to four groups of 50 each, corresponding to semen frozen in 1975, 1976, 1977 and 1978. Fixation with buffered formol saline or buffered gluta- raldehyde was accomplished by pipetting a drop of semen into 4.5 ml vials containing 2 ml of fixative. Wet mounts were prepared by placing a drop of fixed semen on the cen- ter of clean slides under 22 by 30 mm coverslips luted by nail varnish. Semen evaluations were made after thawing and after being placed in a 38°C water bath for 5 hours (Slow Ther- moresistance Test) or in a 45°C water bath for 1 hour (Quick Thermoresistance Test)1. Buffered formol saline material was examined under 1000 x magnifications phase contrast microscope in oil immersion. Differential interference contrast microscopy was used to evaluate wet preparations in buffered glutaraldehyde at 1250 x magnifications in oil immersion. With each method, 200 spermatozoa were evaluated per slide with results give in percentage. Classification into major and minor defects 3 was adopted, besides acrosome evaluation. Data were analysed by analysis of variance8. Differen- ces between treatments were compared using F test, fixing INTRODUCTION brought to you by CORE View metadata, citation and similar papers at core.ac.uk provided by Cadernos Espinosanos (E-Journal)