Cláudia Fernandes Raphael

Effects of bovine sperm cryopreservation using different freezing techniques and cryoprotective agents on plasma, acrosomal and mitochondrial membranes

The success of semen cryopreservation is influenced by several factors, such as freezing curves and cryoprotectants. These two factors are of special interest once they may lead to many important physical‐chemical changes resulting in different degrees of damage in spermatozoa structure. This experiment was designed to compare the effect of bull semen cryopreservation using two freezing techniques: conventional (CT – cooling rate of −0.55 °C min−1 and freezing rate of −19.1 °C min−1) and automated (AT – cooling rate of −0.23 °C min−1 and freezing rate of −15 °C min−1), performed with different curves, and with three cryoprotectants (glycerol, ethylene glycol and dimethyl formamide) on bovine sperm motility and integrity of plasma, acrosomal and mitochondrial membranes. These variables were simultaneously evaluated using the fluorescence probes propidium iodide, fluorescein‐conjugated Pisum sativum agglutinin and MitoTracker Green FM. The effects of freezing techniques, as well as of different cryoprotectants were analysed by the analysis of variance. The means were compared by Fisher’s test. There were no significant differences between freezing techniques (P > 0.05). Glycerol showed higher percentages of motility, vigour and integrity of plasma, acrosomal and mitochondrial membranes than other two cryoprotectants (P < 0.05). Ethylene glycol preserved higher motility and integrity of plasma and mitochondrial membranes than dimethyl formamide (P < 0.05). Sperm motility with glycerol was 30.67 ± 1.41% and 30.50 ± 1.06%, with ethylene glycol was 21.17 ± 1.66% and 21.67 ± 1.13% and with dimethyl formamide was 8.33 ± 0.65% and 9.17 ± 0.72% to CT and AT curves, respectively. The percentage of spermatozoa with simultaneously intact plasma membrane, intact acrosome and mitochondrial function (IPIAH) was 14.82 ± 1.49% (CT) and 15.83 ± 1.26% (AT) to glycerol, 9.20 ± 1.31% (CT) and 9.92 ± 1.29% (AT) to ethylene glycol 4.65 ± 0.93% (CT) and 5.17 ± 0.87% (AT) to dimethyl formamide. Glycerol provided the best results, although nearly 85% of spermatozoa showed some degree of injury in their membranes, suggesting that further studies are required to improve the results of cryopreservation of bovine semen.

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.