Induction of the acrosome reaction test to in vitro estimate embryo production in Nelore cattle

The effectiveness of induction of the acrosome reaction (AR) test as a parameter to in vitro estimate embryo production (IVP) in Nelore breed and the AR pattern by the Trypan Blue/Giemsa (TB) stain were evaluated. Frozen semen samples from ten Nelore bulls were submitted to AR induction and were also evaluated for cleavage and blastocyst rates. The treatments utilized for AR induction were: control (TALP medium), TH (TALP medium + 10μg heparin), TL (TALP medium + 100μg lysophosphatidylcholine) and THL (TALP medium + 10μg heparin + 100μg lysophosphatidylcholine). Sperm acrosomal status and viability were evaluated by TB staining at 0 and after 4h incubation at 38°C. The results obtained for AR presented a significant difference (P<0.05) in the percentage of acrosome reacted live sperm after 4h of incubation in the treatments that received heparin. The cleavage and blastocyst rates were 60% and 38% respectively and a significant difference was observed among bulls (P<0.05). It was founded a satisfactory model to estimate the cleavage and blastocyst rates by AR induction test. Therefore, it can be concluded that the induction of the AR test is a valuable tool to predict the IVP in Nelore breed

Damage assessment of the equine sperm membranes by fluorimetric technique

To validate a practical technique of simultaneous evaluation of the plasma, acrosomal and mitochondrial membranes in equine spermatozoa three fluorescent probes (PI, FITC-PSA and MITO) were associated. Four ejaculates from three stallions (n=12) were diluted in TALP medium and split into 2 aliquots, 1 aliquot was flash frozen in liquid nitrogen to induce damage in cellular membranes. Three treatments were prepared with the following fixed ratios of fresh semen: flash frozen semen: 100:0 (T100), 50:50 (T50), and 0:100 (T0). A 150-µL aliquot of diluted semen of each treatment was added of 2 µL of PI, 2 µL of MITO and 80 µL of FITC-PSA; incubated at 38.5ºC/8 min, and sperm cells were evaluated by epifluorescent microscopy. Based in regression analysis, this could be an efficient and practical technique to assess damage in equine spermatozoa, as it was able to determine the sperm percentage more representative of the potential to fertilize the oocyte.<br>Para validar uma técnica prática de avaliação simultânea das membranas plasmática, acrossomal e mitocondrial em espermatozóides eqüinos três sondas fluorescentes (PI, FITC-PSA e MITO) foram associadas. Quatro ejaculados de três garanhões (n=12) foram diluídos em meio TALP e divididos em duas alíquotas, uma alíquota foi submetida a flash frozen em nitrogênio líquido para induzir danos nas membranas celulares. Três tratamentos foram preparados com as seguintes proporções de sêmen fresco: sêmen flash frozen: 100:0 (T100), 50:50 (T50), e 0:100 (T0). Uma amostra de 150 µL de sêmen diluído de cada tratamento foi adicionada de 2 µL de PI, 2 µL de MITO e 80 µL de FITC-PSA; incubadas à 38,5ºC/8 min, e as células espermáticas foram avaliadas por microscopia de epifluorescência. Baseados na análise de regressão esta é uma técnica eficiente e prática para determinar danos em espermatozóides eqüinos, capaz de determinar a porcentagem de espermatozóides mais representativa do potencial fertilizante do ovócito