ABSTRACT
Este estudo descreveu as características seminais, da membrana plasmática e do acrossoma de espermatozoide congelado/descongelado de 19 ejaculados de garanhões da raça Nordestina. Os aspectos analisados incluíram os parâmetros físicos do sêmen fresco; a motilidade e a longevidade do sêmen diluído e descongelado; a morfologia espermática, integridade funcional e estrutural da membrana plasmática do espermatozoide e a habilidade de ligação do espermatozoide à membrana perivitelina da gema do ovo de galinha do sêmen descongelado. As variáveis foram avaliadas pela ANOVA com post hoc teste de Student Newman-Keuls (P<0,05). A MT e a MP foram maiores (P<0,05) no sêmen diluído do que no descongelado. A percentagem média de defeitos maiores, menores e totais foi muito inferior ao limite recomendado pelo CBRA. A porcentagem de reativos ao HOST foi de 14,21±1,12% e a porcentagem média de membranas íntegras detectadas pelo teste supravital de 62,22±9,06% e pela sonda SYBR-14 de 81,47±26,90. O número médio de espermatozoides ligados à MPV após a descongelação do sêmen foi de 230,39±57,09. A MT e MP no tempo 0 min do TTR foi superior (P<0,05) em relação a 150 min, não diferindo nos tempos 10 min e 30 min. Os resultados demonstram que a utilização dos testes laboratoriais adicionais ajudam no processo de avaliação das amostras, possibilitando a obtenção de informações mais confiáveis e precisas. Embora a criopreservação tenha provocado queda na motilidade seminal, o uso de diluidor contendo amidas minimizou os danos osmóticos nas células espermáticas e manteve a integridade morfológica, funcional e estrutural da membrana plasmática do espermatozoide. Estes resultados são um referencial em estudos futuros uma vez que, inexistem dados comparativos nesta raça...
This paper describes the seminal characteristics of the plasma membrane of frozen-thawed sperm. Nineteen ejaculates of Nordestino horse breed. The aspects analyzed in the physical parameters of fresh semen were total and progressive motility and your longevity after dilution or thawed; sperm morphology, functional and structural integrity of the plasma membrane of the sperm and the sperm-binding ability to the perivitelline membrane of the yolk (MPV) after thawed. The variables were assessed by ANOVA with post hoc test of Student Newman-Keuls test (P<0.05). The total and progressive motility were higher in diluted semen than thawed (P<0.05). The average percentage of the major, minor and total defects was lower than the limit recommended by the CBRA. The percentage of reactive to hypo-osmotic swelling test was 14.21±1.12%, the intact membrane detected by supravitally test was 62.22±9.06% and the SYBR-14 was 81.47±26.9. The ability of sperm to bind to the MPV after thawing semen was 230.39±57.09. The total and progressive motility at time 0 min of termo resistance test was higher than to 150 minutes (P<0.05), and no difference was observed in the times 10 and 30 minutes. The results demonstrate that the use of additional laboratory tests help in the process of evaluation of samples, making possible to obtain more reliable and accurate information. Although cryopreservation has caused decrease in sperm motility and was used diluents with amides to diluted and cryopreservation protocol and this minimized the osmotic damage to sperm cells and maintained the morphological, functional and structural integrity of the plasma membrane of the sperm. These results are a reference for future studies since there are no comparative data on this breed...
Subject(s)
Animals , Male , Horses/physiology , Spermatozoa/physiology , Semen Analysis/veterinary , Cryopreservation/veterinary , Sperm Motility/physiology , Semen Preservation/adverse effects , Semen Preservation/veterinaryABSTRACT
En la aplicación de técnicas reproductivas es importante determinar in vitro la capacidad fecundante de los espermatozoides, para ello se utilizan combinaciones de tinciones para evaluar los diferentes parámetros de función espermática, aumentando así la precisión de la estimación de la muestra. El objetivo del estudio fue comparar la efectividad de la utilización de los fluorocromos 6-CFDA y SYBR-14 combinados con PI para determinar la viabilidad e integridad de la membrana plasmática por citometría de flujo. Se utilizó semen fresco de caninos (n=5) de raza Chihuahua, con una concentración espermática >150x106 esp/ml y motilidad progresiva >80%. Tres protocolos fueron ensayados: grupo 1: SYBR-14/PI, grupo 2: 6-CFDA/PI y grupo 3: PI. La integridad de la membrana plasmática de los espermatozoides fue similar entre grupos 1 y 2, independiente del fluorocromo utilizado (37,26±13,9 y 33,8±14,6, respectivamente; p=0,4601). Asimismo, la viabilidad espermática entre los grupos 1, 2 y 3 (62,7±13,9, 66,1±14,6 y 66,4±13,3, respectivamente; p=0,8987). En conclusión, no se evidenció diferencias en la efectividad para determinar la viabilidad e integridad de la membrana plasmática mediante la utilización de SYBR-14 y 6-CFDA, ambas tinciones pueden ser incorporadas al análisis de rutina de semen canino de raza Chihuahua.
In applying reproductive techniques in vitro it is important to determine the fertilizing capacity of the sperm, for this a combination of dyes were used to assess different parameters of sperm function, thereby increasing the accuracy of the estimation of the sample. In dogs (Canis lupus familiaris) Chihuahua breed there is no precedent for evaluating sperm function parameters. The aim was to assess the viability and plasmatic membrane integrity, basic parameters of sperm function. Propidium iodide (PI) was used, a fluorescent dye-specific DNA, which combined with fluorochromes permeable acts as marker of the sperm membrane integrity. The effectiveness of the use of 6-CFDA and SYBR-14 fluorochromes combined with PI was also compared to determine viability and sperm membrane integrity using flow cytometry. Fresh semen of dogs (n=5) Chihuahua breed was used with a concentration of >200x106 sp/ml and progressive motility >80%. Three protocols were performed: group 1: SYBR-14/PI, group 2: 6-CFDA/PI and group 3: PI. The plasma membrane integrity of sperm was similar, independent of the fluorophore used between groups 1 and 2 (13.9±37.26 and 33.8±14.6, respectively, p=0.4601). This also applied to sperm viability between groups 1, 2 and 3 (62.7±13.9, 66.1±14.6 and 66.4±13.3, respectively, p=0.8987). No difference was demonstrated in effectiveness to determine the viability and integrity of the sperm membrane using SYBR-14 and 6-CFDA, both dyes can be incorporated in to routine analysis of semen in canine Chihuahua breed.