Utilization of fluorescent probe association for simultaneous assessment of plasmatic, acrosomal, and mitochondrial membranes of rooster spermatozoa

Autor(es)

ECC Celeghini ^[1] ; RP Arruda ^[2] ; R Albuquerque ^[3] ; FHA Silva ^[4] ; DE Faria ^[5] ; AFC Andrade ^[6] ; J Nascimento ^[7] ; CF Raphael ^[8]

Afiliação do(s) autor(es)

^[1] Universidade de São Paulo. Faculdade de Medicina Veterinária e Zootecnia. Departamento de Reprodução Animal

^[2] Universidade de São Paulo. Faculdade de Medicina Veterinária e Zootecnia. Departamento de Reprodução Animal

^[3] USP. FMVZ. Departamento de Nutrição e Produção Animal

^[4] USP. Faculdade de Zootecnia e Engenharia de Alimentos. Departamento de Zootecnia

^[5] USP. Faculdade de Zootecnia e Engenharia de Alimentos. Departamento de Zootecnia

^[6] Universidade de São Paulo. Faculdade de Medicina Veterinária e Zootecnia. Departamento de Reprodução Animal

^[7] Universidade de São Paulo. Faculdade de Medicina Veterinária e Zootecnia. Departamento de Reprodução Animal

^[8] Universidade de São Paulo. Faculdade de Medicina Veterinária e Zootecnia. Departamento de Reprodução Animal

• Tipo de documento: Artigo Científico
• Fonte: Brazilian Journal of Poultry Science; v. 9, n. 3, p. 143-149, 2007-09-00.

Resumo

This experiment was designed with the objective of developing a simple, practical, and high repeatability technique for the simultaneous evaluation of the integrity of the plasmatic and acrosomal membranes, as well as funcional mitochondria of domestic fowl spermatozoa using an association of fluorescent probes. Four ejaculates (motility > 80% and abnormal morphology < 10%) from each of six Ross male broiler breeder (n=24) were diluted in TALP sperm medium (25x10(6) spermatozoa/mL) and split into two aliquots, and one of these aliquots was flash frozen in liquid nitrogen and thawed to damage all cellular membranes. Three treatments were prepared from these aliquots, with the following ratios of Fresh semen:Flash frozen semen: 100:0 (T100), 50:50 (T50), and 0:100 (T0). A 150-µL aliquot of diluted semen was placed in a microcentrifuge tube with the addition of 2-µL PI, 2-µL MITO, and 50-µL FITC-PSA, and incubated at 38.5º C/8 min in the dark. An 8-µL sample was placed on a slide, coverslipped, and examined by epifluorescence microscopy. Each sample had 200 cells counted and classified based on the fluorescence emitted by each probe. By regression analysis, plasma membrane integrity, as detected by PI, was determined as: v=4.17+0.82X (R²=0.95). Acrosome integrity, as detected by FITC-PSA, generated the equation: v=4.19+0.84X (R²=0.96). Functional mitochondria was estimated by the equation v=3.20+0.83X (R²=0.96). This is an efficient technique to simultaneously evaluate plasmatic, acrosomal, and mitochondrial membranes in fowl sperm. It is suggested that its application in flow cytometry systems allows this methodology to be applied in large scale. (AU)

• Processo FAPESP: 00/14653-6 - Avaliação dos efeitos da criopreservação do sêmen bovino quanto à motilidade, viabilidade espermática, integridade acrossômica e função mitocondrial determinadas por citometria de fluxo
• Beneficiário: Eneiva Carla Carvalho Celeghini
• Modalidade de apoio: Bolsas no Brasil - Doutorado