RESUMO
This experiment was performed to develop and validate practical techniques for simultaneous evaluation of the integrity of plasma and acrosomal membranes, as well as mitochondrial function in bovine spermatozoa using associations of fluorescent probes. Four protocols of fluorescent probes association were defined: protocol 1: propidium iodide (PI), fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (FITC-PSA) and rhodamine 123; protocol 2: PI, FITC-PSA and MitoTracker Green FM (MITO); protocol 3: PI, Hoechst 33342 (H342), FITC-PSA and CMXRos; and protocol 4: PI, H342, FITC-PSA and JC-1. Three ejaculates from each of the four bulls (n = 12) were utilized, showing sperm motility >/=80% and abnormal morphology =10%. The semen was diluted in Modified Tyrode's medium (TALP) (25 x 10(6) spermatozoa/ml) and split into two aliquots, one sample was flash-frozen in liquid nitrogen and thawed. Samples for three treatments were prepared with the following ratio of fresh semen : flash-frozen semen: 100 : 0, 50 : 50 and 0 : 100. Samples were stained in all four protocols and evaluated by epifluorescence microscopy. Protocol 1 did not result in a satisfactory stain, so it could not be validated. Protocols 2, 3 and 4 were validated and showed high determination coefficient to plasma membrane integrity (R(2) = 0.95, 0.93 and 0.92, respectively), acrosome integrity (R(2) = 0.95, 0.92 and 0.91, respectively) and mitochondrial function (R(2) = 0.84, 0.93 and R(2) = 0.93, respectively). These techniques are efficient for the simultaneous integrity evaluation of plasma and acrosomal membranes and mitochondrial function in bovine spermatozoa. However, JC-1 has an advantage over MITO and CMXRos, as it separates two cell populations with high and low mitochondrial membrane potential.
Assuntos
Acrossomo/fisiologia , Bovinos/fisiologia , Membrana Celular/fisiologia , Membranas Mitocondriais/fisiologia , Espermatozoides/fisiologia , Acrossomo/ultraestrutura , Animais , Membrana Celular/ultraestrutura , Fluoresceína-5-Isotiocianato , Fluorescência , Masculino , Microscopia de Fluorescência/métodos , Microscopia de Fluorescência/veterinária , Membranas Mitocondriais/ultraestrutura , Propídio , Motilidade dos Espermatozoides , Espermatozoides/anormalidades , Espermatozoides/ultraestruturaRESUMO
The purpose of this study was to validate a technique for simultaneous evaluation of the plasma, acrosomal and mitochondrial membranes in boar spermatozoa, using an association of fluorescent probes: Propidium iodide (PI), fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (FITC-PSA) and JC-1. Three ejaculates from each of four different boars, all showing motility >or=80% and abnormal morphology Assuntos
Acrossomo/fisiologia
, Mitocôndrias/metabolismo
, Motilidade dos Espermatozoides/fisiologia
, Espermatozoides/fisiologia
, Suínos/fisiologia
, Acrossomo/ultraestrutura
, Animais
, Membrana Celular/fisiologia
, Membrana Celular/ultraestrutura
, Fluoresceína-5-Isotiocianato
, Fluorescência
, Masculino
, Microscopia de Fluorescência/veterinária
, Mitocôndrias/fisiologia
, Mitocôndrias/ultraestrutura
, Propídio
, Espermatozoides/anormalidades