Practical Techniques for Bovine Sperm Simultaneous Fluorimetric Assessment of Plasma, Acrosomal and Mitochondrial Membranes

Abstract

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.

Efeitos da disponibilidade de sombra a campo sobre características reprodutivas de touros da raça Nelore (Bos indicus) criados na região Sudeste do Brasil

“…An alteration in spermatogenesis microenvironment can lead to greater reactive oxygen species production which alters sperm membrane stability (AITKEN; BAKER, 1995). If microenvironment changes occurred in seminiferous tubules in animals without shaded areas, certainly there would be an increase in sperm with damage in one of the cellular compartments, which would be detectable by the technique described by Celeghini et al (2007). Nichi et al (2006) suggest that Bos indicus individuals have greater protection against lipid peroxidation generated by thermal stress compared with Bos taurus.…”

Identification of ultrastructural and functional damages in sperm from six-banded armadillos (Euphractus sexcinctus) due to cryopreservation

“…Next, the samples were evaluated with an epifluorescence microscope (Episcopic Fluorescent Attachment "EFA" Halogen Lamp Set, Leica, Kista, Sweden), and 200 spermatozoa (per sample) were evaluated for the plasma membrane integrity using PI/H342 association and for mitochondrial membrane potential through CMXRos. Sperm heads marked in blue (H342) were considered to have intact membrane and those totally or partially marked with red (PI) were considered to be not intact; region of the midpiece marked in red was considered as presenting mitochondrial activity (Celeghini et al 2007). …”

“…Immediately after dilution and thawing, a semen aliquot (10µL) was incubated at 34°C for 10 min in a solution composed of the following combination of fluorescent probes: 2µL Propidium Iodide (PI); 0.5mg/ml NaCl at 0.9%; 5µL; CMXRos (Mito Tracker Red®, Molecular Probes, F-7512) at 500nM (dilution of 50µg in 94μL of DPBS); and 3μL Hoechst 342 (H342) (diluted at 25mg/mL in DMSO), according to the methodology described by Celeghini et al (2007). Next, the samples were evaluated with an epifluorescence microscope (Episcopic Fluorescent Attachment "EFA" Halogen Lamp Set, Leica, Kista, Sweden), and 200 spermatozoa (per sample) were evaluated for the plasma membrane integrity using PI/H342 association and for mitochondrial membrane potential through CMXRos.…”

“…Based on this aspect, great emphasis has been given to the use of fluorescent markers that show specific cellular structures and clearly detect the structural and functional integrity of sperm (Celeghini et al 2007).…”

Assessment of different in vitro sperm challenges and in vivo fertility of bovine semen batches

“…In the present study, PI (Sigma,28, was utilized for assessment of plasma membrane integrity, according to adaptations of the protocol proposed by Celeghini et al (2007).…”