E. C. C. Celeghini

Addition of seminal plasma to post-thawing equine semen: what is the effect on sperm cell viability?

Effect of seminal plasma addition after thawing on viability or cryocapacitation is not definitively established. This experiment was performed to verify the effect of adding seminal plasma, autologous or homologous (from an animal with good semen freezability). Five ejaculates from each of four stallions with proven fertility were collected and cryopreserved. The semen was subsequently thawed and divided into the following three treatment groups: no seminal plasma addition after semen thawing (NOSP); the addition of homologous seminal plasma after semen thawing (HSP) and the addition of autologous seminal plasma after semen thawing (ASP). The addition of 20% of seminal plasma led to an increase in the cell population that simultaneously show plasma and acrosomal membrane integrity (p < 0.05). The addition of seminal plasma did not alter the total motility, the amount of cells with mitochondrial membrane potential or the sperm velocities (average path velocity, straight-line velocity and curvilinear velocity). However, the beat/cross-frequency, straightness and linearity were reduced in ASP and HSP groups (p < 0.05). Unexpectedly, the addition of homologous seminal plasma reduced the proportion of cells with progressive motility (p < 0.05) and the addition of autologous seminal plasma reduced the amplitude of the lateral head displacement (p < 0.05). Based on the increase in the cell populations that had the plasma and acrosomal membrane integrity simultaneously identified in this study, we proposed that the addition of seminal plasma (autologous or homologous) into post-thawed semen before insemination could increase semen fertility.

Simultaneous assessment of plasmatic, acrosomal, and mitochondrial membranes in ram sperm by fluorescent probes

Neste experimento, foi definida uma combinacao de sondas fluorescentes: iodeto de propidio (PI), aglutinina de Pisum sativum conjugada ao isotiocionato de fluoresceina (FITC-PSA) e JC-1. Para esta proposta, quatro ejaculados de tres carneiros (n=12), que apresentavam motilidade >80% e alteracoes morfologicas <10%, foram diluidos em meio TALP e divididos em duas aliquotas. Uma aliquota foi submetida a tres ciclos de flash frozen e descongelacao, para induzir danos nas membranas celulares e disturbios na funcao mitocondrial. Tres tratamentos foram preparados com as seguintes proporcoes preestabelecidas de semen fresco: semen submetido a flash frozen: 0:100 (T0), 50:50 (T50) e 100:0 (T100). As amostras foram coradas no protocolo proposto e avaliadas por microscopia de epifluorescencia. Para integridade de membrana plasmatica, detectada pela sonda PI, foi obtida a equacao: v=1,09+0,86X (R2=0,98). O acrossomo intacto, verificado pela sonda FITC-PSA, produziu a equacao: v=2,76+0,92X (R2=0,98). O alto potencial de membrana mitocondrial, marcada em vermelho-alaranjado pelo JC-1, foi estimado pela equacao: v=1,90+0,90X (R2=0,98). As equacoes lineares resultantes demonstraram que a tecnica e eficiente e pratica para avaliacao simultânea das membranas plasmatica, acrossomal e mitocondrial em espermatozoides de carneiros.

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

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 38.5 o C/8 min in the dark. An 8µL 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 (R 2 =0.95). Acrosome integrity, as detected by FITC-PSA, generated the equation: v=4.19+0.84X (R 2 =0.96). Functional mitochondria was estimated by the equation v=3.20+0.83X (R 2 =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.