Evaluation of acrosomal status and sperm viability in fresh and cryopreserved specimens by the use of fluorescent peanut agglutinin lectin in conjunction with hypo-osmotic swelling test

OBJECTIVE: In this study, we evaluated whether the hypo-osmotic swelling test (HOST) can be used as a vital marker in combination with peanut agglutinin (PNA) - labeling in fresh and cryopreserved spermatozoa. MATERIALS AND METHODS: Human sperm populations were exposed to a hypo-osmotic medium for 60 minutes, and then incubated in a 1 µg/mL solution of the fluorescent dye Hoescht 33258 (H33258) for 10 minutes. Excess stain was removed by washing in phosphate-buffered saline (PBS) solution, and the pellet was resuspended in 100 µL of culture medium. Twenty microliters of this solution were subsequently smeared on a microscope slide, and fixed in ice-cold methanol to permeabilize the sperm membranes. The fixed smears were finally incubated in a 40-µg/mL FITC-PNA solution for 20 minutes. Simultaneous assessment of acrosome and viability scores was done in a fluorescent microscope equipped with appropriate filters and phase contrast illumination. The same slide was examined for FITC-PNA labeling, tail swelling, and for Hoechst-33258 staining by interchanging the filters and phase contrast optics. RESULTS: In fresh specimens, HOST was found to provide viability assessments comparable to those obtained using the H33258 method (r = 0.95). However, the results of HOST and H33258 were not correlated in cryopreserved specimens (r = 0.22). There was no alteration of PNA-labeling due to the HOST or H33258. CONCLUSIONS: FITC-PNA labeling in conjunction with the visualization of the morphological change induced by exposure to hypo-osmotic solution provides a simple but effective method for establishing the state of acrosomal membrane and viability in fresh human spermatozoa, but this technique is not reliable for cryopreserved ones.

Efeito de três diferentes diluidores sobre o sêmen canino submetido a dois protocolos de descongelamento / Effect of three different diluent on canine semen submitted to two thawing protocols

Com objetivo de avaliar o efeito de dois protocolos de descongelação do sêmen canino congelado em três diluidores foram coletados ejaculados de nove cães. Cada ejaculado foi dividido em 3 amostras, centrifugado e os pellets tratados sob diferentes protocolos: 1°- meio à base de glicina e gema de ovo; 2° - meio à base de TRIS e 3° - meio M9. Foram envasadas 28 palhetas de O,5ml segundo cada protocolo. Amostras tratadas sob cada protocolo foram avaliadas (M1) quanto a motilidade, vigor e integridade das membranas. As palhetas foram refrigeradas a 5°C por 60 min. e congeladas em N2. Uma amostra de cada protocolo foi avaliada quanto: motilidade, vigor e integridade das membranas após o equilíbrio (M2). Eram sempre descongeladas três pares de palhetas, sendo que cada par havia sido congelado sob um mesmo protocolo. Uma das palhetas de cada par era descongelada a 37°C por 30 segundos e outra a 72°C por 8 segundos. Cada grupo foi avaliado após o descongelamento (M3) quanto a motilidade e vigor espermático, avaliação computadorizada do movimento, integridade das membranas e avaliação acrossomal por meio de FitcPNA e PI. As amostras foram mantidas em banho Maria a 37°C por 1h para o teste de longevidade espermática e reavaliadas (M4) quanto à motilidade e vigor espermáticos, integridade das membranas. Após análise estatística concluímos que as células espermáticas caninas descongeladas a 72°C por 8 segundos apresentaram um maior somatório de bons resultados quanto aos testes de viabilidade espermática in vitro empregados.(AU)

To verify the effect of three different extenders and two thawingtemperatures on frozen-thawed canine sperm characteristics, oneejaculate from nine dogs were separately collected and processed (n=9).Each ejaculate was divided into 3 equal samples and centrifuged. Thepellets were re-suspended using: 1st pellet Glicyne-egg yolk extender(GEY), 2nd pellet - TRIS extender and 3rd pellet - M9 extender and allsamples were filled in 0.5ml straws. A total of 84 straws (28 for eachprotocol) were done. The sperm motility, vigour and plasmamembrane integrity from each protocol were immediately evaluated(M1) and the straws were brought to a refrigerator at 5ºC for 60minutes. After that, a sample from each protocol was warmed up ina water bath 37ºC for 5 min. and sperm motility, vigour and plasmamembrane integrity were evaluated (M2). The straws were frozen inliquid nitrogen. One straw from each protocol was thawed at 37ºCfor 30 sec and another at 72ºC for 8 sec and the sperm motility;vigour, CASA and plasmatic membrane integrity and acrossomalstatus using FITC-PNA stain were evaluated (M3). Plasma membrane,sperm motility and vigour were evaluated after a 1-hour incubation at37ºC (M4). Statistical analysis showed that the higher temperaturehad positive effect on froze-thawed canine sperm characteristics. Thebest results were taken when canine semen was frozen in GEYextender and thawed 72ºC for 8 sec. (AU)